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Sample records for ribosomal gtpase centre

  1. Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs.

    PubMed Central

    Rosendahl, G; Douthwaite, S

    1995-01-01

    The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis. Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite. Twenty point mutations (G-->A and C-->T transitions) and numerous multiple mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability. Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions. These effects are relieved by point mutations at other sites, indicating functional interconnections within the higher order structure of this 23S rRNA region. Several mutations prevent direct binding of r-protein L11 to 23S rRNA in vitro. These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts. Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA. When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the effects of mutations at 1091 and 1099. The proteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometric. Surprisingly, none of the mutations prevents incorporation of L11 into ribosomes in vivo, indicating that other, as yet unidentified, factors are involved in the cooperative assembly process. Images PMID:7630717

  2. The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation

    PubMed Central

    Manikas, Rizos-Georgios; Thomson, Emma; Thoms, Matthias; Hurt, Ed

    2016-01-01

    Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA–protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms. PMID:26823502

  3. The K⁺-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation.

    PubMed

    Manikas, Rizos-Georgios; Thomson, Emma; Thoms, Matthias; Hurt, Ed

    2016-02-29

    Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA-protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms. PMID:26823502

  4. GTPases mechanisms and functions of translation factors on the ribosome.

    PubMed

    Rodnina, M V; Stark, H; Savelsbergh, A; Wieden, H J; Mohr, D; Matassova, N B; Peske, F; Daviter, T; Gualerzi, C O; Wintermeyer, W

    2000-01-01

    The elongation factors (EF) Tu and G and initiation factor 2 (IF2) from bacteria are multidomain GTPases with essential functions in the elongation and initiation phases of translation. They bind to the same site on the ribosome where their low intrinsic GTPase activities are strongly stimulated. The factors differ fundamentally from each other, and from the majority of GTPases, in the mechanisms of GTPase control, the timing of Pi release, and the functional role of GTP hydrolysis. EF-Tu x GTP forms a ternary complex with aminoacyl-tRNA, which binds to the ribosome. Only when a matching codon is recognized, the GTPase of EF-Tu is stimulated, rapid GTP hydrolysis and Pi release take place, EF-Tu rearranges to the GDP form, and aminoacyl-tRNA is released into the peptidyltransferase center. In contrast, EF-G hydrolyzes GTP immediately upon binding to the ribosome, stimulated by ribosomal protein L7/12. Subsequent translocation is driven by the slow dissociation of Pi, suggesting a mechano-chemical function of EF-G. Accordingly, different conformations of EF-G on the ribosome are revealed by cryo-electron microscopy. GTP hydrolysis by IF2 is triggered upon formation of the 70S initiation complex, and the dissociation of Pi and/or IF2 follows a rearrangement of the ribosome into the elongation-competent state. PMID:10937868

  5. The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome

    PubMed Central

    Coatham, Mackenzie L.; Brandon, Harland E.; Fischer, Jeffrey J.; Schümmer, Tobias; Wieden, Hans-Joachim

    2016-01-01

    Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX. PMID:26733579

  6. EF-G-dependent GTPase on the ribosome. conformational change and fusidic acid inhibition.

    PubMed

    Seo, Hyuk-Soo; Abedin, Sameem; Kamp, Detlev; Wilson, Daniel N; Nierhaus, Knud H; Cooperman, Barry S

    2006-02-28

    Protein synthesis studies increasingly focus on delineating the nature of conformational changes occurring as the ribosome exerts its catalytic functions. Here, we use FRET to examine such changes during single-turnover EF-G-dependent GTPase on vacant ribosomes and to elucidate the mechanism by which fusidic acid (FA) inhibits multiple-turnover EF-G.GTPase. Our measurements focus on the distance between the G' region of EF-G and the N-terminal region of L11 (L11-NTD), located within the GTPase activation center of the ribosome. We demonstrate that single-turnover ribosome-dependent EF-G GTPase proceeds according to a kinetic scheme in which rapid G' to L11-NTD movement requires prior GTP hydrolysis and, via branching pathways, either precedes P(i) release (major pathway) or occurs simultaneously with it (minor pathway). Such movement retards P(i) release, with the result that P(i) release is essentially rate-determining in single-turnover GTPase. This is the most significant difference between the EF-G.GTPase activities of vacant and translocating ribosomes [Savelsbergh, A., Katunin, V. I., Mohr, D., Peske, F., Rodnina, M. V., and Wintermeyer, W. (2003) Mol. Cell 11, 1517-1523], which are otherwise quite similar. Both the G' to L11-NTD movement and P(i) release are strongly inhibited by thiostrepton but not by FA. Contrary to the standard view that FA permits only a single round of GTP hydrolysis [Bodley, J. W., Zieve, F. J., and Lin, L. (1970) J. Biol. Chem. 245, 5662-5667], we find that FA functions rather as a slow inhibitor of EF-G.GTPase, permitting a number of GTPase turnovers prior to complete inhibition while inducing a closer approach of EF-G to the GAC than is seen during normal turnover. PMID:16489743

  7. Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome.

    PubMed

    Peske, F; Matassova, N B; Savelsbergh, A; Rodnina, M V; Wintermeyer, W

    2000-08-01

    Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome. PMID:10983996

  8. Ribosome-induced tuning of GTP hydrolysis by a translational GTPase.

    PubMed

    Maracci, Cristina; Peske, Frank; Dannies, Ev; Pohl, Corinna; Rodnina, Marina V

    2014-10-01

    GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that--contrary to current models--the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K(+) ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the γ-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome. PMID:25246550

  9. Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

    PubMed Central

    Maracci, Cristina; Peske, Frank; Dannies, Ev; Pohl, Corinna; Rodnina, Marina V.

    2014-01-01

    GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that—contrary to current models—the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K+ ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the γ-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome. PMID:25246550

  10. Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium

    SciTech Connect

    Nichols, C. E.; Johnson, C.; Lamb, H. K.; Lockyer, M.; Charles, I. G.; Hawkins, A. R.; Stammers, D. K.

    2007-11-01

    The X-ray crystal structure of the GTPase YjeQ from S. typhimurium is presented and compared with those of orthologues from T. maritima and B. subtilis. The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

  11. A Novel Domain in Translational GTPase BipA Mediates Interaction with the 70S Ribosome and Influences GTP Hydrolysis

    SciTech Connect

    deLivron, M.; Makanji, H; Lane, M; Robinson, V

    2009-01-01

    BipA is a universally conserved prokaryotic GTPase that exhibits differential ribosome association in response to stress-related events. It is a member of the translation factor family of GTPases along with EF-G and LepA. BipA has five domains. The N-terminal region of the protein, consisting of GTPase and {beta}-barrel domains, is common to all translational GTPases. BipA domains III and V have structural counterparts in EF-G and LepA. However, the C-terminal domain (CTD) of the protein is unique to the BipA family. To investigate how the individual domains of BipA contribute to the biological properties of the protein, deletion constructs were designed and their GTP hydrolysis and ribosome binding properties assessed. Data presented show that removal of the CTD abolishes the ability of BipA to bind to the ribosome and that ribosome complex formation requires the surface provided by domains III and V and the CTD. Additional mutational analysis was used to outline the BipA-70S interaction surface extending across these domains. Steady state kinetic analyses revealed that successive truncation of domains from the C-terminus resulted in a significant increase in the intrinsic GTP hydrolysis rate and a loss of ribosome-stimulated GTPase activity. These results indicate that, similar to other translational GTPases, the ribosome binding and GTPase activities of BipA are tightly coupled. Such intermolecular regulation likely plays a role in the differential ribosome binding by the protein.

  12. Pre-40S ribosome biogenesis factor Tsr1 is an inactive structural mimic of translational GTPases

    PubMed Central

    McCaughan, Urszula M.; Jayachandran, Uma; Shchepachev, Vadim; Chen, Zhuo Angel; Rappsilber, Juri; Tollervey, David; Cook, Atlanta G.

    2016-01-01

    Budding yeast Tsr1 is a ribosome biogenesis factor with sequence similarity to GTPases, which is essential for cytoplasmic steps in 40S subunit maturation. Here we present the crystal structure of Tsr1 at 3.6 Å. Tsr1 has a similar domain architecture to translational GTPases such as EF-Tu and the selenocysteine incorporation factor SelB. However, active site residues required for GTP binding and hydrolysis are absent, explaining the lack of enzymatic activity in previous analyses. Modelling of Tsr1 into cryo-electron microscopy maps of pre-40S particles shows that a highly acidic surface of Tsr1 is presented on the outside of pre-40S particles, potentially preventing premature binding to 60S subunits. Late pre-40S maturation also requires the GTPase eIF5B and the ATPase Rio1. The location of Tsr1 is predicted to block binding by both factors, strongly indicating that removal of Tsr1 is an essential step during cytoplasmic maturation of 40S ribosomal subunits. PMID:27250689

  13. Pre-40S ribosome biogenesis factor Tsr1 is an inactive structural mimic of translational GTPases.

    PubMed

    McCaughan, Urszula M; Jayachandran, Uma; Shchepachev, Vadim; Chen, Zhuo Angel; Rappsilber, Juri; Tollervey, David; Cook, Atlanta G

    2016-01-01

    Budding yeast Tsr1 is a ribosome biogenesis factor with sequence similarity to GTPases, which is essential for cytoplasmic steps in 40S subunit maturation. Here we present the crystal structure of Tsr1 at 3.6 Å. Tsr1 has a similar domain architecture to translational GTPases such as EF-Tu and the selenocysteine incorporation factor SelB. However, active site residues required for GTP binding and hydrolysis are absent, explaining the lack of enzymatic activity in previous analyses. Modelling of Tsr1 into cryo-electron microscopy maps of pre-40S particles shows that a highly acidic surface of Tsr1 is presented on the outside of pre-40S particles, potentially preventing premature binding to 60S subunits. Late pre-40S maturation also requires the GTPase eIF5B and the ATPase Rio1. The location of Tsr1 is predicted to block binding by both factors, strongly indicating that removal of Tsr1 is an essential step during cytoplasmic maturation of 40S ribosomal subunits. PMID:27250689

  14. Structural insights into the function of a unique tandem GTPase EngA in bacterial ribosome assembly

    PubMed Central

    Zhang, Xiaoxiao; Yan, Kaige; Zhang, Yixiao; Li, Ningning; Ma, Chengying; Li, Zhifei; Zhang, Yanqing; Feng, Boya; Liu, Jing; Sun, Yadong; Xu, Yanji; Lei, Jianlin; Gao, Ning

    2014-01-01

    Many ribosome-interacting GTPases, with proposed functions in ribosome biogenesis, are also implicated in the cellular regulatory coupling between ribosome assembly process and various growth control pathways. EngA is an essential GTPase in bacteria, and intriguingly, it contains two consecutive GTPase domains (GD), being one-of-a-kind among all known GTPases. EngA is required for the 50S subunit maturation. However, its molecular role remains elusive. Here, we present the structure of EngA bound to the 50S subunit. Our data show that EngA binds to the peptidyl transferase center (PTC) and induces dramatic conformational changes on the 50S subunit, which virtually returns the 50S subunit to a state similar to that of the late-stage 50S assembly intermediates. Very interestingly, our data show that the two GDs exhibit a pseudo-two-fold symmetry in the 50S-bound conformation. Our results indicate that EngA recognizes certain forms of the 50S assembly intermediates, and likely facilitates the conformational maturation of the PTC of the 23S rRNA in a direct manner. Furthermore, in a broad context, our data also suggest that EngA might be a sensor of the cellular GTP/GDP ratio, endowed with multiple conformational states, in response to fluctuations in cellular nucleotide pool, to facilitate and regulate ribosome assembly. PMID:25389271

  15. Characterization of the autophosphorylation property of HflX, a ribosome-binding GTPase from Escherichia coli.

    PubMed

    Ghosh, Aditi; Dutta, Dipak; Bandyopadhyay, Kaustav; Parrack, Pradeep

    2016-07-01

    Escherichia coli HflX belongs to the widely distributed but poorly characterized HflX family of translation factor-related GTPases that is conserved from bacteria to humans. A 426-residue polypeptide that binds 50S ribosomes and has both GTPase and ATPase activities, HflX also exhibits autophosphorylation activity. We show that HflX(C), a C-terminal fragment of HflX, has an enhanced autophosphorylation activity compared to the full-length protein. Using a chemical stability assay and thin layer chromatography, we have determined that phosphorylation occurs at a serine residue. Each of the nine serine residues of HflX(C) was mutated to alanine. It was found that all but S211A retained autophosphorylation activity, suggesting that S211, located in the P-loop, was the likely site for autophosphorylation. While the S211A mutant lacked the autophosphorylation site, it possessed strong GTP binding and GTPase activities. PMID:27398305

  16. The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis

    PubMed Central

    Jeon, Young; Park, Yong-Joon; Cho, Hui Kyung; Jung, Hyun Ju; Ahn, Tae-Kyu; Kang, Hunseung; Pai, Hyun-Sook

    2015-01-01

    Nucleostemin is a nucleolar GTP-binding protein that is involved in stem cell proliferation, embryonic development, and ribosome biogenesis in mammals. Plant nucleostemin-like 1 (NSN1) plays a role in embryogenesis, and apical and floral meristem development. In this study, a nucleolar function of NSN1 in the regulation of ribosome biogenesis was identified. Green fluorescent protein (GFP)-fused NSN1 localized to the nucleolus, which was primarily determined by its N-terminal domain. Recombinant NSN1 and its N-terminal domain (NSN1-N) bound to RNA in vitro. Recombinant NSN1 expressed GTPase activity in vitro. NSN1 silencing in Arabidopsis thaliana and Nicotiana benthamiana led to growth retardation and premature senescence. NSN1 interacted with Pescadillo and EBNA1 binding protein 2 (EBP2), which are nucleolar proteins involved in ribosome biogenesis, and with several ribosomal proteins. NSN1, NSN1-N, and EBP2 co-fractionated primarily with the 60S ribosomal large subunit in vivo. Depletion of NSN1 delayed 25S rRNA maturation and biogenesis of the 60S ribosome subunit, and repressed global translation. NSN1-deficient plants exhibited premature leaf senescence, excessive accumulation of reactive oxygen species, and senescence-related gene expression. Taken together, these results suggest that NSN1 plays a crucial role in plant growth and senescence by modulating ribosome biogenesis. PMID:26163696

  17. Antibiotic interactions at the GTPase-associated centre within Escherichia coli 23S rRNA.

    PubMed Central

    Egebjerg, J; Douthwaite, S; Garrett, R A

    1989-01-01

    A comprehensive range of chemical reagents and ribonucleases was employed to investigate the interaction of the antibiotics thiostrepton and micrococcin with the ribosomal protein L11-23S RNA complex and with the 50S subunit. Both antibiotics block processes associated with the ribosomal A-site but differ in their effects on GTP hydrolysis, which is inhibited by thiostrepton and stimulated by micrococcin. The interaction sites of both drugs were shown to occur within the nucleotide sequences A1067-A1098 within the protein L11 binding site on 23S RNA. This region of the ribosome structure is involved in elongation factor-G-dependent GTP hydrolysis and in the stringent response. No effects of drug binding were detected elsewhere in the 23S RNA. In general, the two drugs afforded 23S RNA similar protection from the chemical and nuclease probes in accord with their similar modes of action. One important exception, however, occurred at nucleotide A1067 within a terminal loop where thiostrepton protected the N-1 position while micrococcin rendered it more reactive. This difference correlates with the opposite effects of the two antibiotics on GTPase activity. Images PMID:2470587

  18. The Vibrio harveyi GTPase CgtAV Is Essential and Is Associated with the 50S Ribosomal Subunit

    PubMed Central

    Sikora, A. E.; Zielke, R.; Datta, K.; Maddock, J. R.

    2006-01-01

    It was previously reported that unlike the other obg/cgtA GTPases, the Vibrio harveyi cgtAV is not essential. Here we show that cgtAV was not disrupted in these studies and is, in fact, essential for viability. Depletion of CgtAV did not result in cell elongation. CgtAV is associated with the large ribosomal particle. In light of our results, we predict that the V. harveyi CgtAV protein plays a similar essential role to that seen for Obg/CgtA proteins in other bacteria. PMID:16428430

  19. Nuclear/nucleolar GTPase 2 proteins as a subfamily of YlqF/YawG GTPases function in pre-60S ribosomal subunit maturation of mono- and dicotyledonous plants.

    PubMed

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-03-11

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with k(cat) values 0.12 ± 0.007 min(-1) (n = 6) and 0.087 ± 0.002 min(-1) (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  20. Nuclear/Nucleolar GTPase 2 Proteins as a Subfamily of YlqF/YawG GTPases Function in Pre-60S Ribosomal Subunit Maturation of Mono- and Dicotyledonous Plants*

    PubMed Central

    Im, Chak Han; Hwang, Sung Min; Son, Young Sim; Heo, Jae Bok; Bang, Woo Young; Suwastika, I. Nengah; Shiina, Takashi; Bahk, Jeong Dong

    2011-01-01

    The YlqF/YawG families are important GTPases involved in ribosome biogenesis, cell proliferation, or cell growth, however, no plant homologs have yet to be characterized. Here we isolated rice (Oryza sativa) and Arabidopsis nuclear/nucleolar GTPase 2 (OsNug2 and AtNug2, respectively) that belong to the YawG subfamily and characterized them for pre-60S ribosomal subunit maturation. They showed typical intrinsic YlqF/YawG family GTPase activities in bacteria and yeasts with kcat values 0.12 ± 0.007 min−1 (n = 6) and 0.087 ± 0.002 min−1 (n = 4), respectively, and addition of 60S ribosomal subunits stimulated their activities in vitro. In addition, OsNug2 rescued the lethality of the yeast nug2 null mutant through recovery of 25S pre-rRNA processing. By yeast two-hybrid screening five clones, including a putative one of 60S ribosomal proteins, OsL10a, were isolated. Subcellular localization and pulldown assays resulted in that the N-terminal region of OsNug2 is sufficient for nucleolar/nuclear targeting and association with OsL10a. OsNug2 is physically associated with pre-60S ribosomal complexes highly enriched in the 25S, 5.8S, and 5S rRNA, and its interaction was stimulated by exogenous GTP. Furthermore, the AtNug2 knockdown mutant constructed by the RNAi method showed defective growth on the medium containing cycloheximide. Expression pattern analysis revealed that the distribution of AtNug2 mainly in the meristematic region underlies its potential role in active plant growth. Finally, it is concluded that Nug2/Nog2p GTPase from mono- and didicotyledonous plants is linked to the pre-60S ribosome complex and actively processed 27S into 25S during the ribosomal large subunit maturation process, i.e. prior to export to the cytoplasm. PMID:21205822

  1. TaTypA, a Ribosome-Binding GTPase Protein, Positively Regulates Wheat Resistance to the Stripe Rust Fungus

    PubMed Central

    Liu, Peng; Myo, Thwin; Ma, Wei; Lan, Dingyun; Qi, Tuo; Guo, Jia; Song, Ping; Guo, Jun; Kang, Zhensheng

    2016-01-01

    Tyrosine phosphorylation protein A (TypA/BipA) belongs to the ribosome-binding GTPase superfamily. In many bacterial species, TypA acts as a global stress and virulence regulator and also mediates resistance to the antimicrobial peptide bactericidal permeability-increasing protein. However, the function of TypA in plants under biotic stresses is not known. In this study, we isolated and functionally characterized a stress-responsive TypA gene (TaTypA) from wheat, with three copies located on chromosomes 6A, 6B, and 6D, respectively. Transient expression assays indicated chloroplast localization of TaTypA. The transcript levels of TaTypA were up-regulated in response to treatment with methyl viologen, which induces reactive oxygen species (ROS) in chloroplasts through photoreaction, cold stress, and infection by an avirulent strain of the stripe rust pathogen. Knock down of the expression of TaTypA through virus-induced gene silencing decreased the resistance of wheat to stripe rust accompanied by weakened ROS accumulation and hypersensitive response, an increase in TaCAT and TaSOD expression, and an increase in pathogen hyphal growth and branching. Our findings suggest that TaTypA contributes to resistance in an ROS-dependent manner. PMID:27446108

  2. A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis

    PubMed Central

    Bharat, Amrita; Blanchard, Jan E.; Brown, Eric D.

    2014-01-01

    The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counter-screens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis. PMID:23606650

  3. Review: Translational GTPases.

    PubMed

    Maracci, Cristina; Rodnina, Marina V

    2016-08-01

    Translational GTPases (trGTPases) play key roles in facilitating protein synthesis on the ribosome. Despite the high degree of evolutionary conservation in the sequences of their GTP-binding domains, the rates of GTP hydrolysis and nucleotide exchange vary broadly between different trGTPases. EF-Tu, one of the best-characterized model G proteins, evolved an exceptionally rapid and tightly regulated GTPase activity, which ensures rapid and accurate incorporation of amino acids into the nascent chain. Other trGTPases instead use the energy of GTP hydrolysis to promote movement or to ensure the forward commitment of translation reactions. Recent data suggest the GTPase mechanism of EF-Tu and provide an insight in the catalysis of GTP hydrolysis by its unusual activator, the ribosome. Here we summarize these advances in understanding the functional cycle and the regulation of trGTPases, stimulated by the elucidation of their structures on the ribosome and the progress in dissecting the reaction mechanism of GTPases. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 463-475, 2016. PMID:26971860

  4. rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit▿

    PubMed Central

    Shin, Byung-Sik; Kim, Joo-Ran; Acker, Michael G.; Maher, Kathryn N.; Lorsch, Jon R.; Dever, Thomas E.

    2009-01-01

    The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit. PMID:19029250

  5. Studies on crystal structures, active-centre geometry and depurinating mechanism of two ribosome-inactivating proteins.

    PubMed Central

    Huang, Q; Liu, S; Tang, Y; Jin, S; Wang, Y

    1995-01-01

    Two ribosome-inactivating proteins, trichosanthin and alpha-momorcharin, have been studied in the forms of complexes with ATP or formycin, by an X-ray-crystallographic method at 1.6-2.0 A (0.16-0.20 nm) resolution. The native alpha-momorcharin had been studied at 2.2 A resolution. Structures of trichosanthin were determined by a multiple isomorphous replacement method. Structures of alpha-momorcharin were determined by a molecular replacement method using refined trichosanthin as the searching model. Small ligands in all these complexes have been recognized and built on the difference in electron density. All these structures have been refined to achieve good results, both in terms of crystallography and of ideal geometry. These two proteins show considerable similarity in their three-dimensional folding and to that of related proteins. On the basis of these structures, detailed geometries of the active centres of these two proteins are described and are compared with those of related proteins. In all complexes the interactions between ligand atoms and protein atoms, including hydrophobic forces, aromatic stacking interactions and hydrogen bonds, are found to be specific towards the adenine base. The relationship between the sequence conservation of ribosome-inactivating proteins and their active-centre geometry was analysed. A depurinating mechanism of ribosome-inactivating proteins is proposed on the basis of these results. The N-7 atom of the substrate base group is proposed to be protonated by an acidic residue in the active centre. Images Figure 1 PMID:7619070

  6. Deconstructing ribosome construction

    PubMed Central

    Connolly, Keith; Culver, Gloria

    2013-01-01

    The ribosome is an essential ribonucleoprotein enzyme, and its biogenesis is a fundamental process in all living cells. Recent X-ray crystal structures of the bacterial ribosome and new technologies have allowed a greater interrogation of in vitro ribosome assembly; however, substantially less is known about ribosome biogenesis in vivo. Ongoing investigations are focused on elucidating the cellular processes that facilitate biogenesis of the ribosomal subunits, and many extraribosomal factors, including modification enzymes, remodeling enzymes and GTPases, are being uncovered. Moreover, specific roles for ribosome biogenesis factors in subunit maturation are now being elaborated. Ultimately, such studies will reveal a more complete understanding of processes at work in in vivo ribosome biogenesis. PMID:19376708

  7. Ribosome engineering to promote new crystal forms

    SciTech Connect

    Selmer, Maria; Gao, Yong-Gui; Weixlbaumer, Albert; Ramakrishnan, V.

    2012-05-01

    Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors.

  8. Structural insights into ribosome translocation.

    PubMed

    Ling, Clarence; Ermolenko, Dmitri N

    2016-09-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. Recent structural and single-molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the 'head' domain of small ribosomal subunit undergoes forward- and back-swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF-G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF-G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620-636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  9. Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes.

    PubMed

    Wu, Shan; Tutuncuoglu, Beril; Yan, Kaige; Brown, Hailey; Zhang, Yixiao; Tan, Dan; Gamalinda, Michael; Yuan, Yi; Li, Zhifei; Jakovljevic, Jelena; Ma, Chengying; Lei, Jianlin; Dong, Meng-Qiu; Woolford, John L; Gao, Ning

    2016-06-01

    Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors, organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly. PMID:27251291

  10. Phylogenetic distribution of translational GTPases in bacteria

    PubMed Central

    Margus, Tõnu; Remm, Maido; Tenson, Tanel

    2007-01-01

    Background Translational GTPases are a family of proteins in which GTPase activity is stimulated by the large ribosomal subunit. Conserved sequence features allow members of this family to be identified. Results To achieve accurate protein identification and grouping we have developed a method combining searches with Hidden Markov Model profiles and tree based grouping. We found all the genes for translational GTPases in 191 fully sequenced bacterial genomes. The protein sequences were grouped into nine subfamilies. Analysis of the results shows that three translational GTPases, the translation factors EF-Tu, EF-G and IF2, are present in all organisms examined. In addition, several copies of the genes encoding EF-Tu and EF-G are present in some genomes. In the case of multiple genes for EF-Tu, the gene copies are nearly identical; in the case of multiple EF-G genes, the gene copies have been considerably diverged. The fourth translational GTPase, LepA, the function of which is currently unknown, is also nearly universally conserved in bacteria, being absent from only one organism out of the 191 analyzed. The translation regulator, TypA, is also present in most of the organisms examined, being absent only from bacteria with small genomes. Surprisingly, some of the well studied translational GTPases are present only in a very small number of bacteria. The translation termination factor RF3 is absent from many groups of bacteria with both small and large genomes. The specialized translation factor for selenocysteine incorporation – SelB – was found in only 39 organisms. Similarly, the tetracycline resistance proteins (Tet) are present only in a small number of species. Proteins of the CysN/NodQ subfamily have acquired functions in sulfur metabolism and production of signaling molecules. The genes coding for CysN/NodQ proteins were found in 74 genomes. This protein subfamily is not confined to Proteobacteria, as suggested previously but present also in many other

  11. Ribosome dynamics and the evolutionary history of ribosomes

    NASA Astrophysics Data System (ADS)

    Fox, George E.; Paci, Maxim; Tran, Quyen; Petrov, Anton S.; Williams, Loren D.

    2015-09-01

    The ribosome is a dynamic nanomachine responsible for coded protein synthesis. Its major subsystems were essentially in place at the time of the last universal common ancestor (LUCA). Ribosome evolutionary history thus potentially provides a window into the pre- LUCA world. This history begins with the origins of the peptidyl transferase center where the actual peptide is synthesized and then continues over an extended timeframe as additional functional centers including the GTPase center are added. The large ribosomal RNAs (rRNAs) have grown over time by an accretion process and a model exists that proposes a relative age of each accreted element. We have compared atomic resolution ribosome structures before and after EF-G bound GTP hydrolysis and thereby identified the location of 23 pivot points in the large rRNAs that facilitate ribosome dynamics. Pivots in small subunit helices h28 and h44 appear to be especially central to the process and according to the accretion model significantly older than the other helices containing pivots. Overall, the results suggest that ribosomal dynamics occurred in two phases. In the first phase, an inherently mobile h28/h44 combination provided the flexibility needed to create a dynamic ribosome that was essentially a Brownian machine. This addition likely made coded peptide synthesis possible by facilitating movement of a primitive mRNA. During the second phase, addition of pivoting elements and the creation of a factor binding site allowed the regulation of the inherent motion created by h28/h44. All of these events likely occurred before LUCA.

  12. Modular domains of the Dicistroviridae intergenic internal ribosome entry site

    PubMed Central

    Jang, Christopher J.; Jan, Eric

    2010-01-01

    The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae viral family can directly assemble 80S ribosomes and initiate translation at a non-AUG codon from the ribosomal A-site. These functions are directed by two independently folded domains of the IGR IRES. One domain, composed of overlapping pseudoknots II and III (PKII/III), mediates ribosome recruitment. The second domain, composed of PKI, mimics a tRNA anticodon–codon interaction to position the ribosome at the ribosomal A-site. Although adopting a common secondary structure, the dicistrovirus IGR IRESs can be grouped into two classes based on distinct features within each domain. In this study, we report on the modularity of the IGR IRESs and show that the ribosome-binding domain and the tRNA anticodon mimicry domain are functionally interchangeable between the Type I and the Type II IGR IRESs. Using structural probing, ribosome-binding assays, and ribosome positioning analysis by toeprinting assays, we show that the chimeric IRESs fold properly, assemble 80S ribosomes, and can mediate IRES translation in rabbit reticulocyte lysates. We also demonstrate that the chimeric IRESs can stimulate the ribosome-dependent GTPase activity of eEF2, which suggests that the ribosome is primed for a step downstream from IRES binding. Overall, the results demonstrate that the dicistrovirus IGR IRESs are composed of two modular domains that work in concert to manipulate the ribosome and direct translation initiation. PMID:20423979

  13. Crystal structure of YjeQ from Thermotoga maritima contains a circularly permuted GTPase domain

    PubMed Central

    Shin, Dong Hae; Lou, Yun; Jancarik, Jaru; Yokota, Hisao; Kim, Rosalind; Kim, Sung-Hou

    2004-01-01

    We have determined the crystal structure of the GDP complex of the YjeQ protein from Thermotoga maritima (TmYjeQ), a member of the YjeQ GTPase subfamaily. TmYjeQ, a homologue of Escherichia coli YjeQ, which is known to bind to the ribosome, is composed of three domains: an N-terminal oligonucleotide/oligosaccharide-binding fold domain, a central GTPase domain, and a C-terminal zinc-finger domain. The crystal structure of TmYjeQ reveals two interesting domains: a circularly permutated GTPase domain and an unusual zinc-finger domain. The binding mode of GDP in the GTPase domain of TmYjeQ is similar to those of GDP or GTP analogs in ras proteins, a prototype GTPase. The N-terminal oligonucleotide/oligosaccharide-binding fold domain, together with the GTPase domain, forms the extended RNA-binding site. The C-terminal domain has an unusual zinc-finger motif composed of Cys-250, Cys-255, Cys-263, and His-257, with a remote structural similarity to a portion of a DNA-repair protein, rad51 fragment. The overall structural features of TmYjeQ make it a good candidate for an RNA-binding protein, which is consistent with the biochemical data of the YjeQ subfamily in binding to the ribosome. PMID:15331784

  14. Structural stabilization of GTP-binding domains in circularly permuted GTPases: Implications for RNA binding

    PubMed Central

    Anand, Baskaran; Verma, Sunil Kumar; Prakash, Balaji

    2006-01-01

    GTP hydrolysis by GTPases requires crucial residues embedded in a conserved G-domain as sequence motifs G1–G5. However, in some of the recently identified GTPases, the motif order is circularly permuted. All possible circular permutations were identified after artificially permuting the classical GTPases and subjecting them to profile Hidden Markov Model searches. This revealed G4–G5–G1–G2–G3 as the only possible circular permutation that can exist in nature. It was also possible to recognize a structural rationale for the absence of other permutations, which either destabilize the invariant GTPase fold or disrupt regions that provide critical residues for GTP binding and hydrolysis, such as Switch-I and Switch-II. The circular permutation relocates Switch-II to the C-terminus and leaves it unfastened, thus affecting GTP binding and hydrolysis. Stabilizing this region would require the presence of an additional domain following Switch-II. Circularly permuted GTPases (cpGTPases) conform to such a requirement and always possess an ‘anchoring’ C-terminal domain. There are four sub-families of cpGTPases, of which three possess an additional domain N-terminal to the G-domain. The biochemical function of these domains, based on available experimental reports and domain recognition analysis carried out here, are suggestive of RNA binding. The features that dictate RNA binding are unique to each subfamily. It is possible that RNA-binding modulates GTP binding or vice versa. In addition, phylogenetic analysis indicates a closer evolutionary relationship between cpGTPases and a set of universally conserved bacterial GTPases that bind the ribosome. It appears that cpGTPases are RNA-binding proteins possessing a means to relate GTP binding to RNA binding. PMID:16648363

  15. Ribosomal proteins: functions beyond the ribosome

    PubMed Central

    Zhou, Xiang; Liao, Wen-Juan; Liao, Jun-Ming; Liao, Peng; Lu, Hua

    2015-01-01

    Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation, their ribosome-independent functions have also been greatly appreciated. Over the past decade, more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress. In addition, these ribosomal proteins are involved in various physiological and pathological processes. This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins, as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis, immune signaling, and development. We also propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics. PMID:25735597

  16. Functional characterization of EngA(MS), a P-loop GTPase of Mycobacterium smegmatis.

    PubMed

    Agarwal, Nisheeth; Pareek, Madhu; Thakur, Preeti; Pathak, Vibha

    2012-01-01

    Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngA(MS)) is highly conserved in mycobacteria. Homology modeling of EngA(MS) reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngA(MS) purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngA(MS) protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngA(MS) with 50S subunit of ribosome and specifically C-terminal domains of EngA(MS) are required to facilitate this interaction. Moreover, EngA(MS) devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngA(MS)-50S interaction. PMID:22506030

  17. Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der.

    PubMed

    Hwang, Jihwan; Tseitin, Vladimir; Ramnarayan, Kal; Shenderovich, Mark D; Inouye, Masayori

    2012-05-01

    Der is an essential and widely conserved GTPase that assists assembly of a large ribosomal subunit in bacteria. Der associates specifically with the 50S subunit in a GTP-dependent manner and the cells depleted of Der accumulate the structurally unstable 50S subunit, which dissociates into an aberrant subunit at a lower Mg(2+) concentration. As Der is an essential and ubiquitous protein in bacteria, it may prove to be an ideal cellular target against which new antibiotics can be developed. In the present study, we describe our attempts to identify novel antibiotics specifically targeting Der GTPase. We performed the structure-based design of Der inhibitors using the X-ray crystal structure of Thermotoga maritima Der (TmDer). Virtual screening of commercially available chemical library retrieved 257 small molecules that potentially inhibit Der GTPase activity. These 257 chemicals were tested for their in vitro effects on TmDer GTPase and in vivo antibacterial activities. We identified three structurally diverse compounds, SBI-34462, -34566 and -34612, that are both biologically active against bacterial cells and putative enzymatic inhibitors of Der GTPase homologs. We also presented the possible interactions of each compound with the Der GTP-binding site to understand the mechanism of inhibition. Therefore, our lead compounds inhibiting Der GTPase provide scaffolds for the development of novel antibiotics against antibiotic-resistant pathogenic bacteria. PMID:22377538

  18. Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome.

    PubMed

    Kumar, Veerendra; Ero, Rya; Ahmed, Tofayel; Goh, Kwok Jian; Zhan, Yin; Bhushan, Shashi; Gao, Yong-Gui

    2016-06-17

    Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial. Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution. Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP and P site tRNA. In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution. Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail. PMID:27137929

  19. The activity of the acidic phosphoproteins from the 80 S rat liver ribosome.

    PubMed

    MacConnell, W P; Kaplan, N O

    1982-05-25

    The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes. PMID:6121796

  20. Dynamin, a membrane remodelling GTPase

    PubMed Central

    Ferguson, Shawn M.; De Camilli, Pietro

    2012-01-01

    Dynamin, the founding member of a family of dynamin-like GTPases (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live cell imaging, cell free-studies, X-ray crystallography and genetic studies in mice has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged that highlight specific contributions of this GTPase to the physiology of different tissues. PMID:22233676

  1. A monovalent cation acts as structural and catalytic cofactor in translational GTPases

    PubMed Central

    Kuhle, Bernhard; Ficner, Ralf

    2014-01-01

    Translational GTPases are universally conserved GTP hydrolyzing enzymes, critical for fidelity and speed of ribosomal protein biosynthesis. Despite their central roles, the mechanisms of GTP-dependent conformational switching and GTP hydrolysis that govern the function of trGTPases remain poorly understood. Here, we provide biochemical and high-resolution structural evidence that eIF5B and aEF1A/EF-Tu bound to GTP or GTPγS coordinate a monovalent cation (M+) in their active site. Our data reveal that M+ ions form constitutive components of the catalytic machinery in trGTPases acting as structural cofactor to stabilize the GTP-bound “on” state. Additionally, the M+ ion provides a positive charge into the active site analogous to the arginine-finger in the Ras-RasGAP system indicating a similar role as catalytic element that stabilizes the transition state of the hydrolysis reaction. In sequence and structure, the coordination shell for the M+ ion is, with exception of eIF2γ, highly conserved among trGTPases from bacteria to human. We therefore propose a universal mechanism of M+-dependent conformational switching and GTP hydrolysis among trGTPases with important consequences for the interpretation of available biochemical and structural data. PMID:25225612

  2. Rho GTPases and their effector proteins.

    PubMed Central

    Bishop, A L; Hall, A

    2000-01-01

    Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field. PMID:10816416

  3. Regulating Rho GTPases and their regulators.

    PubMed

    Hodge, Richard G; Ridley, Anne J

    2016-08-01

    Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases. PMID:27301673

  4. Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

    PubMed Central

    Lancaster, Lorna E; Savelsbergh, Andreas; Kleanthous, Colin; Wintermeyer, Wolfgang; Rodnina, Marina V

    2008-01-01

    The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon–anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon–recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA–mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin. PMID:18485067

  5. Deregulation of Rho GTPases in cancer

    PubMed Central

    Porter, Andrew P.; Papaioannou, Alexandra; Malliri, Angeliki

    2016-01-01

    ABSTRACT In vitro and in vivo studies and evidence from human tumors have long implicated Rho GTPase signaling in the formation and dissemination of a range of cancers. Recently next generation sequencing has identified direct mutations of Rho GTPases in human cancers. Moreover, the effects of ablating genes encoding Rho GTPases and their regulators in mouse models, or through pharmacological inhibition, strongly suggests that targeting Rho GTPase signaling could constitute an effective treatment. In this review we will explore the various ways in which Rho signaling can be deregulated in human cancers. PMID:27104658

  6. Ribosome-dependent activation of stringent control.

    PubMed

    Brown, Alan; Fernández, Israel S; Gordiyenko, Yuliya; Ramakrishnan, V

    2016-06-01

    In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics. PMID:27279228

  7. Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement.

    PubMed

    Budkevich, Tatyana V; Giesebrecht, Jan; Behrmann, Elmar; Loerke, Justus; Ramrath, David J F; Mielke, Thorsten; Ismer, Jochen; Hildebrand, Peter W; Tung, Chang-Shung; Nierhaus, Knud H; Sanbonmatsu, Karissa Y; Spahn, Christian M T

    2014-07-01

    The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection. PMID:24995983

  8. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

    PubMed Central

    Li, Lian-Feng; Yu, Jiahui; Li, Yongfeng; Wang, Jinghan; Li, Su; Zhang, Lingkai; Xia, Shui-Li; Yang, Qian; Wang, Xiao; Yu, Shaoxiong; Luo, Yuzi; Sun, Yuan; Zhu, Yan; Munir, Muhammad

    2016-01-01

    ABSTRACT Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV

  9. LRRK2 autophosphorylation enhances its GTPase activity.

    PubMed

    Liu, Zhiyong; Mobley, James A; DeLucas, Lawrence J; Kahn, Richard A; West, Andrew B

    2016-01-01

    The leucine-rich repeat kinase (LRRK)-2 protein contains nonoverlapping GTPase and kinase domains, and mutation in either domain can cause Parkinson disease. GTPase proteins are critical upstream modulators of many effector protein kinases. In LRRK2, this paradigm may be reversed, as the kinase domain phosphorylates its own GTPase domain. In this study, we found that the ameba LRRK2 ortholog ROCO4 phosphorylates the GTPase domain [termed Ras-of-complex (ROC) domain in this family] of human LRRK2 on the same residues as the human LRRK2 kinase. Phosphorylation of ROC enhances its rate of GTP hydrolysis [from kcat (catalytic constant) 0.007 to 0.016 min(-1)], without affecting GTP or GDP dissociation kinetics [koff = 0.093 and 0.148 min(-1) for GTP and GDP, respectively). Phosphorylation also promotes the formation of ROC dimers, although GTPase activity appears to be equivalent between purified dimers and monomers. Modeling experiments show that phosphorylation induces conformational changes at the critical p-loop structure. Finally, ROC appears to be one of many GTPases phosphorylated in p-loop residues, as revealed by alignment of LRRK2 autophosphorylation sites with GTPases annotated in the phosphoproteome database. These results provide an example of a novel mechanism for kinase-mediated control of GTPase activity. PMID:26396237

  10. The ribosome filter redux.

    PubMed

    Mauro, Vincent P; Edelman, Gerald M

    2007-09-15

    The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it. PMID:17890902

  11. Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases.

    PubMed

    Eiler, Daniel; Lin, Jinzhong; Simonetti, Angelita; Klaholz, Bruno P; Steitz, Thomas A

    2013-09-24

    The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S ribosomal initiation complex and stimulates its assembly to the 50S ribosomal subunit to make the 70S ribosome. The 3.1-Å resolution X-ray crystal structures of the full-length Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the "articulated lever" mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with ribosomal complexes. PMID:24029018

  12. Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

    PubMed Central

    Koch, Miriam; Flür, Sara; Kreutz, Christoph; Ennifar, Eric; Micura, Ronald; Polacek, Norbert

    2015-01-01

    Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases. PMID:25941362

  13. Formins as effector proteins of Rho GTPases

    PubMed Central

    Kühn, Sonja; Geyer, Matthias

    2014-01-01

    Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells. PMID:24914801

  14. Potassium Acts as a GTPase-Activating Element on Each Nucleotide-Binding Domain of the Essential Bacillus subtilis EngA

    PubMed Central

    Foucher, Anne-Emmanuelle; Reiser, Jean-Baptiste; Ebel, Christine; Housset, Dominique; Jault, Jean-Michel

    2012-01-01

    EngA proteins form a unique family of bacterial GTPases with two GTP-binding domains in tandem, namely GD1 and GD2, followed by a KH (K-homology) domain. They have been shown to interact with the bacterial ribosome and to be involved in its biogenesis. Most prokaryotic EngA possess a high GTPase activity in contrast to eukaryotic GTPases that act mainly as molecular switches. Here, we have purified and characterized the GTPase activity of the Bacillus subtilis EngA and two shortened EngA variants that only contain GD1 or GD2-KH. Interestingly, the GTPase activity of GD1 alone is similar to that of the whole EngA, whereas GD2-KH has a 150-fold lower GTPase activity. At physiological concentration, potassium strongly stimulates the GTPase activity of each protein construct. Interestingly, it affects neither the affinities for nucleotides nor the monomeric status of EngA or the GD1 domain. Thus, potassium likely acts as a chemical GTPase-activating element as proposed for another bacterial GTPase like MnmE. However, unlike MnmE, potassium does not promote dimerization of EngA. In addition, we solved two crystal structures of full-length EngA. One of them contained for the first time a GTP-like analogue bound to GD2 while GD1 was free. Surprisingly, its overall fold was similar to a previously solved structure with GDP bound to both sites. Our data indicate that a significant structural change must occur upon K+ binding to GD2, and a comparison with T. maritima EngA and MnmE structures allowed us to propose a model explaining the chemical basis for the different GTPase activities of GD1 and GD2. PMID:23056455

  15. Locking GTPases covalently in their functional states

    NASA Astrophysics Data System (ADS)

    Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Müller, Matthias P.; Goody, Roger S.

    2015-07-01

    GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase-acryl-nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.

  16. The ribosomal database project.

    PubMed Central

    Larsen, N; Olsen, G J; Maidak, B L; McCaughey, M J; Overbeek, R; Macke, T J; Marsh, T L; Woese, C R

    1993-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server. PMID:8332524

  17. The Ribosomal Database Project.

    PubMed Central

    Maidak, B L; Larsen, N; McCaughey, M J; Overbeek, R; Olsen, G J; Fogel, K; Blandy, J; Woese, C R

    1994-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services, and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server/rdp.life.uiuc.edu) and gopher (rdpgopher.life.uiuc.edu). The electronic mail server also provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for chimeric nature of newly sequenced rRNAs, and automated alignment. PMID:7524021

  18. The Ribosomal Database Project

    PubMed Central

    Olsen, Gary J.; Overbeek, Ross; Larsen, Niels; Marsh, Terry L.; McCaughey, Michael J.; Maciukenas, Michael A.; Kuan, Wen-Min; Macke, Thomas J.; Xing, Yuqing; Woese, Carl R.

    1992-01-01

    The Ribosomal Database Project (RDP) compiles ribosomal sequences and related data, and redistributes them in aligned and phylogenetically ordered form to its user community. It also offers various software packages for handling, analyzing and displaying sequences. In addition, the RDP offers (or will offer) certain analytic services. At present the project is in an intermediate stage of development. PMID:1598241

  19. Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association.

    PubMed

    Sprink, Thiemo; Ramrath, David J F; Yamamoto, Hiroshi; Yamamoto, Kaori; Loerke, Justus; Ismer, Jochen; Hildebrand, Peter W; Scheerer, Patrick; Bürger, Jörg; Mielke, Thorsten; Spahn, Christian M T

    2016-03-01

    Throughout the four phases of protein biosynthesis-initiation, elongation, termination, and recycling-the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAi (Met) in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation. PMID:26973877

  20. Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

    PubMed Central

    Sprink, Thiemo; Ramrath, David J. F.; Yamamoto, Hiroshi; Yamamoto, Kaori; Loerke, Justus; Ismer, Jochen; Hildebrand, Peter W.; Scheerer, Patrick; Bürger, Jörg; Mielke, Thorsten; Spahn, Christian M. T.

    2016-01-01

    Throughout the four phases of protein biosynthesis—initiation, elongation, termination, and recycling—the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation. PMID:26973877

  1. Ribosomal Dynamics: Intrinsic Instability of a Molecular Machine

    NASA Astrophysics Data System (ADS)

    Gao, Haixiao; Le Barron, Jamie; Frank, Joachim

    Ribosomes are molecular machines that translate genetic message into nascent peptides, through a complex dynamics interplay with mRNAs, tRNAs, and various protein factors. A prominent example of ribosomal dynamics is the rotation of small ribosomal subunit with respect to a large subunit, characterized as the "ratchet motion," which is triggered by the binding of several translation factors. Here, we analyze two kinds of ribosomal ratchet motions, induced by the binding of EF-G and RF3, respectively, as previously observed by cryo-electron microscopy. Using the flexible fitting technique (real-space refinement) and an RNA secondary structure display tool (coloRNA), we obtained quasi-atomic models of the ribosome in these ratchet-motion-related functional states and mapped the observed differences onto the highly conserved RNA secondary structure. Comparisons between two sets of ratchet motions revealed that, while the overall patterns of the RNA displacement are very similar, several local regions stand out in their differential behavior, including the highly conserved GAC (GTPase-associated-center) region. We postulate that these regions are important in modulating general ratchet motion and bestowing it with the dynamic characteristics required for the specific function.

  2. When ribosomes go bad: diseases of ribosome biogenesis

    PubMed Central

    Freed, Emily F.; Bleichert, Franziska; Dutca, Laura M.; Baserga, Susan J.

    2010-01-01

    Ribosomes are vital for cell growth and survival. Until recently, it was believed that mutations in ribosomes or ribosome biogenesis factors would be lethal, due to the essential nature of these complexes. However, in the last few decades, a number of diseases of ribosome biogenesis have been discovered. It remains a challenge in the field to elucidate the molecular mechanisms underlying them. PMID:20174677

  3. Rho GTPase signalling in cell migration

    PubMed Central

    Ridley, Anne J

    2015-01-01

    Cells migrate in multiple different ways depending on their environment, which includes the extracellular matrix composition, interactions with other cells, and chemical stimuli. For all types of cell migration, Rho GTPases play a central role, although the relative contribution of each Rho GTPase depends on the environment and cell type. Here, I review recent advances in our understanding of how Rho GTPases contribute to different types of migration, comparing lamellipodium-driven versus bleb-driven migration modes. I also describe how cells migrate across the endothelium. In addition to Rho, Rac and Cdc42, which are well known to regulate migration, I discuss the roles of other less-well characterized members of the Rho family. PMID:26363959

  4. Are There Rab GTPases in Archaea?

    PubMed Central

    Surkont, Jaroslaw; Pereira-Leal, Jose B.

    2016-01-01

    A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins. PMID:27034425

  5. Are There Rab GTPases in Archaea?

    PubMed

    Surkont, Jaroslaw; Pereira-Leal, Jose B

    2016-07-01

    A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins. PMID:27034425

  6. Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome

    SciTech Connect

    Zhou, Jie; Lancaster, Laura; Trakhanov, Sergei; Noller, Harry F.

    2012-03-26

    The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 {angstrom} crystal structure of the RF3 {center_dot} GDPNP {center_dot} ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7{sup o} rotation of the body and 14{sup o} rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.

  7. Small RAB GTPases Regulate Multiple Steps of Mitosis.

    PubMed

    Miserey-Lenkei, Stéphanie; Colombo, María I

    2016-01-01

    GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis. PMID:26925400

  8. Small RAB GTPases Regulate Multiple Steps of Mitosis

    PubMed Central

    Miserey-Lenkei, Stéphanie; Colombo, María I.

    2016-01-01

    GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis. PMID:26925400

  9. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  10. Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome

    PubMed Central

    Wilden, Berthold; Savelsbergh, Andreas; Rodnina, Marina V.; Wintermeyer, Wolfgang

    2006-01-01

    The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP–GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP/GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 μM range (37°C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF-G·GDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosome-complex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA–mRNA movement, and Pi release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover. PMID:16940356

  11. Approaches of targeting Rho GTPases in cancer drug discovery

    PubMed Central

    Lin, Yuan; Zheng, Yi

    2016-01-01

    Introduction Rho GTPases are master regulators of actomyosin structure and dynamics and play pivotal roles in a variety of cellular processes including cell morphology, gene transcription, cell cycle progression and cell adhesion. Because aberrant Rho GTPase signaling activities are widely associated with human cancer, key components of Rho GTPase signaling pathways have attracted increasing interest as potential therapeutic targets. Similar to Ras, Rho GTPases themselves were, until recently, deemed “undruggable” because of structure-function considerations. Several approaches to interfere with Rho GTPase signaling have been explored and show promise as new ways for tackling cancer cells. Areas covered This review focuses on the recent progress in targeting the signaling activities of three prototypical Rho GTPases, i.e. RhoA, Rac1, and Cdc42. The authors describe the involvement of these Rho GTPases, their key regulators and effectors in cancer. Furthermore, the authors discuss the current approaches for rationally targeting aberrant Rho GTPases along their signaling cascades, upstream and downstream of Rho GTPases and posttranslational modifications at a molecular level. Expert opinion To date, while no clinically effective drugs targeting Rho GTPase signaling for cancer treatment are available, tool compounds and lead drugs that pharmacologically inhibit Rho GTPase pathways have shown promise. Small molecule inhibitors targeting Rho GTPase signaling may add new treatment options for future precision cancer therapy, particularly in combination with other anti-cancer agents. PMID:26087073

  12. The ribosome returned

    PubMed Central

    Moore, Peter B

    2009-01-01

    Since the mid-1990s, insights obtained from electron microscopy and X-ray crystallography have transformed our understanding of how the most important ribozyme in the cell, the ribosome, catalyzes protein synthesis. This review provides a brief account of how this structural revolution came to pass, and the impact it has had on our understanding of how the ribosome decodes messenger RNAs. PMID:19222865

  13. Ribosome-omics of the human ribosome

    PubMed Central

    Gupta, Varun; Warner, Jonathan R.

    2014-01-01

    The torrent of RNA-seq data becoming available not only furnishes an overview of the entire transcriptome but also provides tools to focus on specific areas of interest. Our focus on the synthesis of ribosomes asked whether the abundance of mRNAs encoding ribosomal proteins (RPs) matched the equimolar need for the RPs in the assembly of ribosomes. We were at first surprised to find, in the mapping data of ENCODE and other sources, that there were nearly 100-fold differences in the level of the mRNAs encoding the different RPs. However, after correcting for the mapping ambiguities introduced by the presence of more than 2000 pseudogenes derived from RP mRNAs, we show that for 80%–90% of the RP genes, the molar ratio of mRNAs varies less than threefold, with little tissue specificity. Nevertheless, since the RPs are needed in equimolar amounts, there must be sluggish or regulated translation of the more abundant RP mRNAs and/or substantial turnover of unused RPs. In addition, seven of the RPs have subsidiary genes, three of which are pseudogenes that have been “rescued” by the introduction of promoters and/or upstream introns. Several of these are transcribed in a tissue-specific manner, e.g., RPL10L in testis and RPL3L in muscle, leading to potential variation in ribosome structure from one tissue to another. Of the 376 introns in the RP genes, a single one is alternatively spliced in a tissue-specific manner. PMID:24860015

  14. Invited review: Small GTPases and their GAPs.

    PubMed

    Mishra, Ashwini K; Lambright, David G

    2016-08-01

    Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bound inactive states under control of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which accelerate slow intrinsic rates of activation by nucleotide exchange and deactivation by GTP hydrolysis, respectively. Here we review developments leading to current understanding of intrinsic and GAP catalyzed GTP hydrolytic reactions in small GTPases from structural, molecular and chemical mechanistic perspectives. Despite the apparent simplicity of the GTPase cycle, the structural bases underlying the hallmark hydrolytic reaction and catalytic acceleration by GAPs are considerably more diverse than originally anticipated. Even the most fundamental aspects of the reaction mechanism have been challenging to decipher. Through a combination of experimental and in silico approaches, the outlines of a consensus view have begun to emerge for the best studied paradigms. Nevertheless, recent observations indicate that there is still much to be learned. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 431-448, 2016. PMID:26972107

  15. Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome

    PubMed Central

    Åqvist, Johan; Kamerlin, Shina C.L.

    2015-01-01

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (TΔS‡) amounts to more than 7 kcal/mol at 25 °C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s−1. This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle. PMID:26497916

  16. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins

    PubMed Central

    Gamalinda, Michael; Woolford, John L

    2015-01-01

    The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders. PMID:26779413

  17. An Allosteric Pathway Revealed in the Ribosome Binding Stress Factor BipA

    SciTech Connect

    Makanji, H.; deLivron, M; Robinson, V

    2009-01-01

    BipA is a highly conserved prokaryotic GTPase that functions as a master regulator of stress and virulence processes in bacteria. It is a member of the translational factor family of GTPases along with EF-G, IF-2 and LepA. Structural and biochemical data suggest that ribosome binding specificity for each member of this family lies in an effector domain. As with other bacterial GTPases, the ribosome binding and GTPase activities of this protein are tightly coupled. However, the mechanism by which this occurs is still unknown. A series of experiments have been designed to probe structural features of the protein to see if we can pinpoint specific areas of BipA, perhaps even individual residues, which are important to its association with the ribosome. Included in the list are the C-terminal effector domain of the protein, which is distinct to the BipA family of proteins, and amino acid residues in the switch I and II regions of the G domain. Using sucrose density gradients, we have shown that the C-terminal domain is required in order for BipA to bind to the ribosome. Moreover, deletion of this domain increases the GTP hydrolysis rates of the protein, likely through relief of inhibitory contacts. Additional evidence has revealed an allosteric connection between the conformationally flexible switch II region and the C-terminal domain of BipA. Site directed mutagenesis, sucrose gradients and malachite green assays are being used to elucidate the details of this coupling.

  18. Alignment/misalignment hypothesis for tRNA selection by the ribosome.

    PubMed

    Sanbonmatsu, K Y

    2006-08-01

    Transfer RNAs (tRNAs) are the adaptor molecules that allow the ribosome to decode genetic information during protein synthesis. During decoding, the ribosome must chose the tRNA whose anticodon corresponds to the codon inscribed in the messenger RNA to incorporate the correct amino acid into the growing polypeptide chain. Fidelity is improved dramatically by a GTP hydrolysis event. Information about the correctness of the anticodon must be sent from the decoding center to the elongation factor, EF-Tu, where the GTP hydrolysis takes place. A second discrimination event entails the accommodation of the aminoacyl-tRNA into its fully bound A/A state inside the ribosome. Here, we present a hypothesis for a specific mechanism of signal transduction through the tRNA, which operates during GTPase activation and accommodation. We propose that the rigidity of the tRNA plays an important role in the transmission of the decoding signal. While the tRNA must flex during binding and accommodation, its anisotropic stiffness enables precise positioning of the acceptor arm in the A/T state, the A/A state and the accommodation corridor. Correct alignment will result in optimal GTPase activation and accommodation rates. Incorrect tRNAs, however, whose anticodons are misaligned, will also have acceptor arms that are misaligned, resulting in sub-optimal GTPase activation and accommodation rates. In the case of GTPase activation, it is possible that the misalignment of the acceptor arm affects the rate directly, by altering the conformational change of the switch region of EF-Tu, or indirectly, by changing the alignment of EF-Tu with respect to the sarcin-ricin loop (SRL) of the large ribosomal subunit. PMID:16890341

  19. A Pan-GTPase Inhibitor as a Molecular Probe

    PubMed Central

    Hong, Lin; Guo, Yuna; BasuRay, Soumik; Agola, Jacob O.; Romero, Elsa; Simpson, Denise S.; Schroeder, Chad E.; Simons, Peter; Waller, Anna; Garcia, Matthew; Carter, Mark; Ursu, Oleg; Gouveia, Kristine; Golden, Jennifer E.; Aubé, Jeffrey; Wandinger-Ness, Angela; Sklar, Larry A.

    2015-01-01

    Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed. PMID:26247207

  20. Crystallography of ribosomal particles

    NASA Astrophysics Data System (ADS)

    Yonath, A.; Frolow, F.; Shoham, M.; Müssig, J.; Makowski, I.; Glotz, C.; Jahn, W.; Weinstein, S.; Wittmann, H. G.

    1988-07-01

    Several forms of three-dimensional crystals and two-dimensional sheets of intact ribosomes and their subunits have been obtained as a result of: (a) an extensive systematic investigation of the parameters involved in crystallization, (b) a development of an experimental procedure for controlling the volumes of the crystallization droplets, (c) a study of the nucleation process, and (d) introducing a delicate seeding procedure coupled with variations in the ratios of mono- and divalent ions in the crystallization medium. In all cases only biologically active particles could be crystallized, and the crystalline material retains its integrity and activity. Crystallographic data have been collected from crystals of 50S ribosomal subunits, using synchrotron radiation at temperatures between + 19 and - 180°C. Although at 4°C the higher resolution reflections decay within minutes in the synchrotron beam, at cryo-temperature there was hardly any radiation damage, and a complete set of data to about 6Åresolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50S ribosomal subunits from a mutant of B. stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with in the native ones. Models, aimed to be used for low resolution phasing, have been reconstructed from two-dimensional sheets of 70S ribosomes and 50S subunits at 47 and 30Å, respectively. These models show the overall structure of these particles, the contact areas between the large and small subunits, the space where protein synthesis might take place and a tunnel which may provide the path for the nascent protein chain.

  1. The Ribosome Comes Alive

    PubMed Central

    2010-01-01

    This essay is a reflection on the ways the X-ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo-EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo-EM and single-particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample (“story in a sample”), providing snapshots of an entire subprocess of translation, such as translocation or decoding. PMID:21072331

  2. The Ribosome Comes Alive.

    PubMed

    Frank, Joachim

    2010-06-18

    This essay is a reflection on the ways the X-ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo-EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo-EM and single-particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample ("story in a sample"), providing snapshots of an entire subprocess of translation, such as translocation or decoding. PMID:21072331

  3. YsxC, an essential protein in Staphylococcus aureus crucial for ribosome assembly/stability

    PubMed Central

    2009-01-01

    Background Bacterial growth and division requires a core set of essential proteins, several of which are still of unknown function. They are also attractive targets for the development of new antibiotics. YsxC is a member of a family of GTPases highly conserved across eubacteria with a possible ribosome associated function. Results Here, we demonstrate by the creation of a conditional lethal mutant that ysxC is apparently essential for growth in S. aureus. To begin to elucidate YsxC function, a translational fusion of YsxC to the CBP-ProteinA tag in the staphylococcal chromosome was made, enabling Tandem Affinity Purification (TAP) of YsxC-interacting partners. These included the ribosomal proteins S2, S10 and L17, as well as the β' subunit of the RNA polymerase. YsxC was then shown to copurify with ribosomes as an accessory protein specifically localizing to the 50 S subunit. YsxC depletion led to a decrease in the presence of mature ribosomes, indicating a role in ribosome assembly and/or stability in S. aureus. Conclusions In this study we demonstrate that YsxC of S. aureus localizes to the ribosomes, is crucial for ribosomal stability and is apparently essential for the life of S. aureus. PMID:20021644

  4. Rag GTPase in amino acid signaling.

    PubMed

    Kim, Joungmok; Kim, Eunjung

    2016-04-01

    Rag small GTPases were identified as the sixth subfamily of Ras-related GTPases. Compelling evidence suggests that Rag heterodimer (RagA/B and RagC/D) plays an important role in amino acid signaling toward mechanistic target of rapamycin complex 1 (mTORC1), which is a central player in the control of cell growth in response to a variety of environmental cues, including growth factors, cellular energy/oxygen status, and amino acids. Upon amino acid stimulation, active Rag heterodimer (RagA/B(GTP)-RagC/D(GDP)) recruits mTORC1 to the lysosomal membrane where Rheb resides. In this review, we provide a current understanding on the amino acid-regulated cell growth control via Rag-mTORC1 with recently identified key players, including Ragulator, v-ATPase, and GATOR complexes. Moreover, the functions of Rag in physiological systems and in autophagy are discussed. PMID:26781224

  5. Small GTPases as regulators of cell division

    PubMed Central

    Militello, Rodrigo; Colombo, María I.

    2013-01-01

    The superfamily of small GTPases serves as a signal transducer to regulate a diverse array of cellular functions. The members of this superfamily are structurally and functionally classified into at least 5 groups (Ras, Rho/Rac, Rab, Arf, and Ran) and they are involved in the control of cell proliferation and differentiation, regulation of the actin cytoskeleton, membrane trafficking, and nuclear transport. It is widely reported that members of the Rab family participate in the control of intracellular membrane trafficking through the interaction with specific effector molecules. However, many Rabs and other small GTPases have also been shown to function in cell division. In this review, we discuss current knowledge about Rab proteins regulating different stages of the cell cycle, such as the congregation and segregation of chromosomes (during metaphase) and the final stage of cell division known as cytokinesis, in which a cell is cleaved originating 2 daughter cells. PMID:24265858

  6. A tale of two GTPases in cotranslational protein targeting

    PubMed Central

    Saraogi, Ishu; Akopian, David; Shan, Shu-Ou

    2011-01-01

    Guanosine triphosphatases (GTPases) comprise a superfamily of proteins that provide molecular switches to regulate numerous cellular processes. The “GTPase switch” paradigm, in which a GTPase acts as a bimodal switch that is turned “on” and “off” by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases. Recent work on a pair of GTPases in the signal recognition particle (SRP) pathway has revealed a distinct mode of GTPase regulation. Instead of the classical GTPase switch, the two GTPases in the SRP and SRP receptor undergo a series of conformational changes during their dimerization and reciprocal activation. Each conformational rearrangement provides a point at which these GTPases can communicate with and respond to their upstream and downstream biological cues, thus ensuring the spatial and temporal precision of all the molecular events in the SRP pathway. We suggest that the SRP and SRP receptor represent an emerging class of “multistate” regulatory GTPases uniquely suited to provide exquisite control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion. PMID:21898651

  7. Locking GTPases covalently in their functional states

    PubMed Central

    Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Müller, Matthias P.; Goody, Roger S.

    2015-01-01

    GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase–acryl–nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins. PMID:26178622

  8. Ribosome-inactivating proteins

    PubMed Central

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms. PMID:24071927

  9. Rho GTPases at the crossroad of signaling networks in mammals

    PubMed Central

    Wojnacki, José; Quassollo, Gonzalo; Marzolo, María-Paz; Cáceres, Alfredo

    2014-01-01

    Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization. PMID:24691223

  10. Ribosome Assembly as Antimicrobial Target.

    PubMed

    Nikolay, Rainer; Schmidt, Sabine; Schlömer, Renate; Deuerling, Elke; Nierhaus, Knud H

    2016-01-01

    Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors. PMID:27240412

  11. Ribosome Assembly as Antimicrobial Target

    PubMed Central

    Nikolay, Rainer; Schmidt, Sabine; Schlömer, Renate; Deuerling, Elke; Nierhaus, Knud H.

    2016-01-01

    Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors. PMID:27240412

  12. Ribosomal Database Project II

    DOE Data Explorer

    The Ribosomal Database Project (RDP) provides ribosome related data and services to the scientific community, including online data analysis and aligned and annotated Bacterial small-subunit 16S rRNA sequences. As of March 2008, RDP Release 10 is available and currently (August 2009) contains 1,074,075 aligned 16S rRNA sequences. Data that can be downloaded include zipped GenBank and FASTA alignment files, a histogram (in Excel) of the number of RDP sequences spanning each base position, data in the Functional Gene Pipeline Repository, and various user submitted data. The RDP-II website also provides numerous analysis tools.[From the RDP-II home page at http://rdp.cme.msu.edu/index.jsp

  13. Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome

    PubMed Central

    Borg, Anneli; Pavlov, Michael; Ehrenberg, Måns

    2016-01-01

    The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance. PMID:27001509

  14. Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement

    PubMed Central

    Budkevich, Tatyana V.; Giesebrecht, Jan; Behrmann, Elmar; Loerke, Justus; Ramrath, David J.F.; Mielke, Thorsten; Ismer, Jochen; Hildebrand, Peter W.; Tung, Chang-Shung; Nierhaus, Knud H.; Sanbonmatsu, Karissa Y.; Spahn, Christian M.T.

    2014-01-01

    SUMMARY The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present sub-nanometer resolution cryo-electron microscopy maps of the mammalian 80S ribosome in the post-translocational state and in complex with the eukaryotic eEF1A•Val-tRNA•GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the post-translocational state from the classical pre-translocational state ribosome. We term this motion “subunit rolling”. Correspondingly, a mammalian decoding complex visualized in sub-states before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection. PMID:24995983

  15. Ribosome recycling induces optimal translation rate at low ribosomal availability.

    PubMed

    Marshall, E; Stansfield, I; Romano, M C

    2014-09-01

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired. PMID:25008084

  16. Ribosome recycling induces optimal translation rate at low ribosomal availability

    PubMed Central

    Marshall, E.; Stansfield, I.; Romano, M. C.

    2014-01-01

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3′ end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called ‘closed-loop’ model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired. PMID:25008084

  17. Regulation of phagocytosis by Rho GTPases

    PubMed Central

    Mao, Yingyu; Finnemann, Silvia C

    2015-01-01

    Phagocytosis is defined as a cellular uptake pathway for particles of greater than 0.5 μm in diameter. Particle clearance by phagocytosis is of critical importance for tissue health and homeostasis. The ultimate goal of anti-pathogen phagocytosis is to destroy engulfed bacteria or fungi and to stimulate cell-cell signaling that mount an efficient immune defense. In contrast, clearance phagocytosis of apoptotic cells and cell debris is anti-inflammatory. High capacity clearance phagocytosis pathways are available to professional phagocytes of the immune system and the retina. Additionally, a low capacity, so-called bystander phagocytic pathway is available to most other cell types. Different phagocytic pathways are stimulated by particle ligation of distinct surface receptors but all forms of phagocytosis require F-actin recruitment beneath tethered particles and F-actin re-arrangement promoting engulfment, which are controlled by Rho family GTPases. The specificity of Rho GTPase activity during the different forms of phagocytosis by mammalian cells is the subject of this review. PMID:25941749

  18. Ribosomes in a Stacked Array

    PubMed Central

    Yamashita, Yui; Kadokura, Yoshitomo; Sotta, Naoyuki; Fujiwara, Toru; Takigawa, Ichigaku; Satake, Akiko; Onouchi, Hitoshi; Naito, Satoshi

    2014-01-01

    Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state. PMID:24652291

  19. Exploring Ribosome Positioning on Translating Transcripts with Ribosome Profiling.

    PubMed

    Spealman, Pieter; Wang, Hao; May, Gemma; Kingsford, Carl; McManus, C Joel

    2016-01-01

    Recent technological advances (e.g., microarrays and massively parallel sequencing) have facilitated genome-wide measurement of many aspects of gene regulation. Ribosome profiling is a high-throughput sequencing method used to measure gene expression at the level of translation. This is accomplished by quantifying both the number of translating ribosomes and their locations on mRNA transcripts. The inventors of this approach have published several methods papers detailing its implementation and addressing the basics of ribosome profiling data analysis. Here we describe our lab's procedure, which differs in some respects from those published previously. In addition, we describe a data analysis pipeline, Ribomap, for ribosome profiling data. Ribomap allocates sequence reads to alternative mRNA isoforms, normalizes sequencing bias along transcripts using RNA-seq data, and outputs count vectors of per-codon ribosome occupancy for each transcript. PMID:26463378

  20. Dual use of GTP hydrolysis by elongation factor G on the ribosome

    PubMed Central

    Cunha, Carlos E.; Belardinelli, Riccardo; Peske, Frank; Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2013-01-01

    Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor. PMID:26824016

  1. Dual use of GTP hydrolysis by elongation factor G on the ribosome.

    PubMed

    Cunha, Carlos E; Belardinelli, Riccardo; Peske, Frank; Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V

    2013-01-01

    Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor. PMID:26824016

  2. Salmonella Enterica Serovar Typhimurium BipA Exhibits Two Distinct Ribosome Binding Modes

    SciTech Connect

    deLivron, M.; Robinson, V

    2008-01-01

    BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess the GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.

  3. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    SciTech Connect

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  4. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    NASA Astrophysics Data System (ADS)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-01

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  5. Small GTPase regulation of GPCR anterograde trafficking

    PubMed Central

    Wang, Guansong; Wu, Guangyu

    2011-01-01

    The physiological functions of heterotrimeric G protein-coupled receptors (GPCRs) are dictated by their intracellular trafficking and precise targeting to the functional destinations. Over the past decades, most studies on the trafficking of GPCRs have focused on the events involved in endocytosis and recycling. In contrast, the molecular mechanisms underlying anterograde transport of newly synthesized GPCRs from the endoplasmic reticulum (ER) to the cell surface have just begun to be revealed. In this review, we will discuss current advances in understanding the role of Ras-like GTPases, specifically the Rab and Sar1/ARF subfamilies, in regulating cell-surface transport of GPCRs en route from the ER and the Golgi. PMID:22015208

  6. Solubilization and partial characterization of a microsomal high affinity GTPase

    SciTech Connect

    Nicchitta, C.; Williamson, J.R.

    1987-05-01

    Isolated rat liver microsomes release sequestered Ca/sup 2 +/ following addition of GTP. In contrast to permeabilized cells, GTP dependent microsomal Ca/sup 2 +/ release requires low concentrations of polyethylene glycol (PEG). They have identified a microsomal, PEG-sensitive high affinity GTPase which shares a number of characteristics with the GTP-dependent Ca/sup 2 +/ release system. To aid in further characterization of this activity they have initiated studies on the solubilization and purification of the microsomal GTPases. When microsomes are solubilized under the following conditions (150 mM NaCl, 5 mg protein/ml, 1% Triton X-114) PEG sensitive GTPase activity selectively partitions into the detergent rich phase of the Triton X-114 extract. As observed in intact microsomal membranes the Triton X-114 soluble GTPase is maximally stimulated by 3% PEG. Half maximal stimulation is observed at 1% PEG. PEG increases the Vmax of this activity; no effects on Km were observed. The Km for GTP of the detergent soluble GTPase is 5 ..mu..M. This GTPase is sensitive to inhibition by sulfhydryl reagents. PEG-sensitive GTPase activity was completely inhibited in the presence of 25 ..mu..M p-hydroxymercuribenzoate (PHMB); half maximal inhibition was observed at 5 ..mu..M. Labeling of the Triton X-114 extract with the photosensitive compound (/sup 32/P) 8-azido GTP indicated the presence of two prominent GTP binding proteins of approximate molecular weights 17 and 54 kD.

  7. Interferon-Inducible GTPases in Host Resistance, Inflammation and Disease.

    PubMed

    Pilla-Moffett, Danielle; Barber, Matthew F; Taylor, Gregory A; Coers, Jörn

    2016-08-28

    Cell-autonomous immunity is essential for host organisms to defend themselves against invasive microbes. In vertebrates, both the adaptive and the innate branches of the immune system operate cell-autonomous defenses as key effector mechanisms that are induced by pro-inflammatory interferons (IFNs). IFNs can activate cell-intrinsic host defenses in virtually any cell type ranging from professional phagocytes to mucosal epithelial cells. Much of this IFN-induced host resistance program is dependent on four families of IFN-inducible GTPases: the myxovirus resistance proteins, the immunity-related GTPases, the guanylate-binding proteins (GBPs), and the very large IFN-inducible GTPases. These GTPase families provide host resistance to a variety of viral, bacterial, and protozoan pathogens through the sequestration of microbial proteins, manipulation of vesicle trafficking, regulation of antimicrobial autophagy (xenophagy), execution of intracellular membranolytic pathways, and the activation of inflammasomes. This review discusses our current knowledge of the molecular function of IFN-inducible GTPases in providing host resistance, as well as their role in the pathogenesis of autoinflammatory Crohn's disease. While substantial advances were made in the recent past, few of the known functions of IFN-inducible GTPases have been explored in any depth, and new functions await discovery. This review will therefore highlight key areas of future exploration that promise to advance our understanding of the role of IFN-inducible GTPases in human diseases. PMID:27181197

  8. Timing Is Everything: GTPase Regulation in Phototransduction

    PubMed Central

    Arshavsky, Vadim Y.; Wensel, Theodore G.

    2013-01-01

    As the molecular mechanisms of vertebrate phototransduction became increasingly clear in the 1980s, a persistent problem was the discrepancy between the slow GTP hydrolysis catalyzed by the phototransduction G protein, transducin, and the much more rapid physiological recovery of photoreceptor cells from light stimuli. Beginning with a report published in 1989, a series of studies revealed that transducin GTPase activity could approach the rate needed to explain physiological recovery kinetics in the presence of one or more factors present in rod outer segment membranes. One by one, these factors were identified, beginning with PDEγ, the inhibitory subunit of the cGMP phosphodiesterase activated by transducin. There followed the discovery of the crucial role played by the regulator of G protein signaling, RGS9, a member of a ubiquitous family of GTPase-accelerating proteins, or GAPs, for heterotrimeric G proteins. Soon after, the G protein β isoform Gβ5 was identified as an obligate partner subunit, followed by the discovery or R9AP, a transmembrane protein that anchors the RGS9 GAP complex to the disk membrane, and is essential for the localization, stability, and activity of this complex in vivo. The physiological importance of all of the members of this complex was made clear first by knockout mouse models, and then by the discovery of a human visual defect, bradyopsia, caused by an inherited deficiency in one of the GAP components. Further insights have been gained by high-resolution crystal structures of subcomplexes, and by extensive mechanistic studies both in vitro and in animal models. PMID:24265205

  9. Synchronous tRNA movements during translocation on the ribosome are orchestrated by elongation factor G and GTP hydrolysis.

    PubMed

    Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V

    2014-10-01

    The translocation of tRNAs through the ribosome proceeds through numerous small steps in which tRNAs gradually shift their positions on the small and large ribosomal subunits. The most urgent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase elongation factor G (EF-G), promotes directed movement; and (iii) how the energy of GTP hydrolysis is coupled to movement. In the light of recent advances in biophysical and structural studies, we argue that intermediate states of translocation are snapshots of dynamic fluctuations that guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the energy regimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms. PMID:25118068

  10. Evolutionary analyses of the 12-kDa acidic ribosomal P-proteins reveal a distinct protein of higher plant ribosomes

    PubMed Central

    Szick, Kathleen; Springer, Mark; Bailey-Serres, Julia

    1998-01-01

    The P-protein complex of eukaryotic ribosomes forms a lateral stalk structure in the active site of the large ribosomal subunit and is thought to assist in the elongation phase of translation by stimulating GTPase activity of elongation factor-2 and removal of deacylated tRNA. The complex in animals, fungi, and protozoans is composed of the acidic phosphoproteins P0 (35 kDa), P1 (11–12 kDa), and P2 (11–12 kDa). Previously we demonstrated by protein purification and microsequencing that ribosomes of maize (Zea mays L.) contain P0, one type of P1, two types of P2, and a distinct P1/P2 type protein designated P3. Here we implemented distance matrices, maximum parsimony, and neighbor-joining analyses to assess the evolutionary relationships between the 12 kDa P-proteins of maize and representative eukaryotic species. The analyses identify P3, found to date only in mono- and dicotyledonous plants, as an evolutionarily distinct P-protein. Plants possess three distinct groups of 12 kDa P-proteins (P1, P2, and P3), whereas animals, fungi, and protozoans possess only two distinct groups (P1 and P2). These findings demonstrate that the P-protein complex has evolved into a highly divergent complex with respect to protein composition despite its critical position within the active site of the ribosome. PMID:9482893

  11. Analysis of the Small GTPase Gene Superfamily of Arabidopsis1

    PubMed Central

    Vernoud, Vanessa; Horton, Amy C.; Yang, Zhenbiao; Nielsen, Erik

    2003-01-01

    Small GTP-binding proteins regulate diverse processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. These proteins function as molecular switches that cycle between “active” and “inactive” states, and this cycle is linked to the binding and hydrolysis of GTP. The Arabidopsis genome contains 93 genes that encode small GTP-binding protein homologs. Phylogenetic analysis of these genes shows that plants contain Rab, Rho, Arf, and Ran GTPases, but no Ras GTPases. We have assembled complete lists of these small GTPases families, as well as accessory proteins that control their activity, and review what is known of the functions of individual members of these families in Arabidopsis. We also discuss the possible roles of these GTPases in relation to their similarity to orthologs with known functions and localizations in yeast and/or animal systems. PMID:12644670

  12. Rag GTPases are cardioprotective by regulating lysosomal function

    PubMed Central

    Kim, Young Chul; Mo, Jung-Soon; Jewell, Jenna L.; Russell, Ryan C.; Wu, Xiaohui; Sadoshima, Junichi; Guan, Kun-Liang

    2014-01-01

    The Rag family proteins are Ras-like small GTPases that play a critical role in amino acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here, we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised due to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection. PMID:24980141

  13. Exploring potassium-dependent GTP hydrolysis in TEES family GTPases.

    PubMed

    Rafay, Abu; Majumdar, Soneya; Prakash, Balaji

    2012-01-01

    GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a 'K-loop' present in switch-I, were suggested as attributes of GTPases employing a K(+)-mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K(+)-activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases - Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) - and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K(+)-mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K(+)-mechanism in GTPases and hence warrant extensive structural investigations. PMID:23650596

  14. Reverse engineering GTPase programming languages with reconstituted signaling networks.

    PubMed

    Coyle, Scott M

    2016-07-01

    The Ras superfamily GTPases represent one of the most prolific signaling currencies used in Eukaryotes. With these remarkable molecules, evolution has built GTPase networks that control diverse cellular processes such as growth, morphology, motility and trafficking. (1-4) Our knowledge of the individual players that underlie the function of these networks is deep; decades of biochemical and structural data has provided a mechanistic understanding of the molecules that turn GTPases ON and OFF, as well as how those GTPase states signal by controlling the assembly of downstream effectors. However, we know less about how these different activities work together as a system to specify complex dynamic signaling outcomes. Decoding this molecular "programming language" would help us understand how different species and cell types have used the same GTPase machinery in different ways to accomplish different tasks, and would also provide new insights as to how mutations to these networks can cause disease. We recently developed a bead-based microscopy assay to watch reconstituted H-Ras signaling systems at work under arbitrary configurations of regulators and effectors. (5) Here we highlight key observations and insights from this study and propose extensions to our method to further study this and other GTPase signaling systems. PMID:27128855

  15. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu

    PubMed Central

    Agirrezabala, Xabier; Frank, Joachim

    2010-01-01

    The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 Å, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G – GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation. PMID:20025795

  16. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu.

    PubMed

    Agirrezabala, Xabier; Frank, Joachim

    2009-08-01

    The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 A, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G - GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation. PMID:20025795

  17. The C-terminal α-helix of YsxC is essential for its binding to 50S ribosome and rRNAs.

    PubMed

    Wicker-Planquart, Catherine; Ceres, Nicoletta; Jault, Jean-Michel

    2015-07-22

    YsxC is an essential P-loop GTPase that interacts with the 50S subunit of the ribosome. The putative implication in ribosome binding of two basic clusters of YsxC, a conserved positively charged patch including R31, R116, H117 and K146 lying adjacent to the nucleotide-binding site, and the C-terminal alpha helix, was investigated. C-terminal truncation variants of YsxC were unable to bind to both ribosome and rRNAs, whereas mutations in the other cluster did not affect YsxC binding. Our results indicate that the basic C-terminal region of YsxC is required for its binding to the 50S ribosomal subunit. PMID:26103561

  18. The mechanics of ribosomal translocation.

    PubMed

    Achenbach, John; Nierhaus, Knud H

    2015-07-01

    The ribosome translates the sequence of codons of an mRNA into the corresponding sequence of amino acids as it moves along the mRNA with a codon-step width of about 10 Å. The movement of the million-dalton complex ribosome is triggered by the universal elongation factor G (EF2 in archaea and eukaryotes) and is termed translocation. Unraveling the molecular details of translocation is one of the most challenging tasks of current ribosome research. In the last two years, enormous progress has been obtained by highly-resolved X-ray and cryo-electron microscopic structures as well as by sophisticated biochemical approaches concerning the trigger and control of the movement of the tRNA2·mRNA complex inside the ribosome during translocation. This review inspects and surveys these achievements. PMID:25514765

  19. The Ribosomal Database Project (RDP).

    PubMed Central

    Maidak, B L; Olsen, G J; Larsen, N; Overbeek, R; McCaughey, M J; Woese, C R

    1996-01-01

    The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and World Wide Web (WWW)(http://rdpwww.life.uiuc.edu/). The electronic mail and WWW servers provide ribosomal probe checking, screening for possible chimeric rRNA sequences, automated alignment and approximate phylogenetic placement of user-submitted sequences on an existing phylogenetic tree. PMID:8594608

  20. RAC/ROP GTPases and auxin signaling.

    PubMed

    Wu, Hen-ming; Hazak, Ora; Cheung, Alice Y; Yalovsky, Shaul

    2011-04-01

    Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity and may also undertake some Ras functions. Here, we discuss the advances made over the last decade that implicate RAC/ROPs as mediators for auxin-regulated gene expression, rapid cell surface-located auxin signaling, and directional auxin transport. We also describe experimental data indicating that auxin-RAC/ROP crosstalk may form regulatory feedback loops and theoretical modeling that attempts to connect local auxin gradients with RAC/ROP regulation of cell polarity. We hope that by discussing these experimental and modeling studies, this perspective will stimulate efforts to further refine our understanding of auxin signaling via the RAC/ROP molecular switch. PMID:21478442

  1. The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3; end of 16S rRNA

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tarasov, Sergey G.; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2012-03-26

    Era, composed of a GTPase domain and a K homology domain, is essential for bacterial cell viability. It is required for the maturation of 16S rRNA and assembly of the 30S ribosomal subunit. We showed previously that the protein recognizes nine nucleotides (1531{sup AUCACCUCC}1539) near the 3{prime} end of 16S rRNA, and that this recognition stimulates GTP-hydrolyzing activity of Era. In all three kingdoms of life, the 1530{sup GAUCA}1534 sequence and helix 45 (h45) (nucleotides 1506-1529) are highly conserved. It has been shown that the 1530{sup GA}1531 to 1530{sup AG}1531 double mutation severely affects the viability of bacteria. However, whether Era interacts with G1530 and/or h45 and whether such interactions (if any) contribute to the stimulation of Era's GTPase activity were not known. Here, we report two RNA structures that contain nucleotides 1506-1542 (RNA301), one in complex with Era and GDPNP (GNP), a nonhydrolysable GTP-analogue, and the other in complex with Era, GNP, and the KsgA methyltransferase. The structures show that Era recognizes 10 nucleotides, including G1530, and that Era also binds h45. Moreover, GTPase assay experiments show that G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the intrinsic GTPase activity of Era, its interaction with Era is important for binding and is essential for the protein to function, leading to the discovery of a new cold-sensitive phenotype of Era.

  2. ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

    PubMed Central

    Corrigan, Rebecca M.; Bellows, Lauren E.; Wood, Alison

    2016-01-01

    The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases—RsgA, RbgA, Era, HflX, and ObgE—as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria. PMID:26951678

  3. Insights into RNA binding by the anticancer drug cisplatin from the crystal structure of cisplatin-modified ribosome.

    PubMed

    Melnikov, Sergey V; Söll, Dieter; Steitz, Thomas A; Polikanov, Yury S

    2016-06-01

    Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin-RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome-the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity. PMID:27079977

  4. [Ribosomal RNA Evolution

    NASA Technical Reports Server (NTRS)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  5. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    PubMed Central

    Poirot, Olivier; Timsit, Youri

    2016-01-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing. PMID:27225526

  6. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome.

    PubMed

    Poirot, Olivier; Timsit, Youri

    2016-01-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through "molecular synapses", ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the "sensory-proteins" innervate the functional ribosomal sites, while the "inter-proteins" interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing. PMID:27225526

  7. The Caulobacter crescentus CgtAC Protein Cosediments with the Free 50S Ribosomal Subunit

    PubMed Central

    Lin, Bin; Thayer, Desiree A.; Maddock, Janine R.

    2004-01-01

    The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtAC protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtAC does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtAC to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtAC. Assays of epitope-tagged wild-type and mutant variants of CgtAC indicate that the C terminus of CgtAC is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtAC alleles to function in vivo. Depletion of CgtAC led to perturbations in the polysome profile, raising the possibility that CgtAC is involved in ribosome assembly or stability. PMID:14702318

  8. Mechanism of eIF6 release from the nascent 60S ribosomal subunit.

    PubMed

    Weis, Félix; Giudice, Emmanuel; Churcher, Mark; Jin, Li; Hilcenko, Christine; Wong, Chi C; Traynor, David; Kay, Robert R; Warren, Alan J

    2015-11-01

    SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias. PMID:26479198

  9. Mechanism of eIF6 release from the nascent 60S ribosomal subunit

    PubMed Central

    Weis, Félix; Giudice, Emmanuel; Churcher, Mark; Jin, Li; Hilcenko, Christine; Wong, Chi C; Traynor, David; Kay, Robert R; Warren, Alan J

    2016-01-01

    SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS–EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias. PMID:26479198

  10. Correlated conformational events in EF-G and the ribosome regulate translocation

    PubMed Central

    Munro, James B.; Wasserman, Michael R.; Altman, Roger B.; Wang, Leyi; Blanchard, Scott C.

    2010-01-01

    In bacteria, the translocation of tRNA and mRNA with respect to the ribosome is catalyzed by the conserved GTPase, elongation factor-G (EF-G). In order to probe the rate determining features in this process, EF-G-catalyzed translocation was imaged from two unique structural perspectives using single-molecule fluorescence resonance energy transfer. The data reveal that the rate at which the ribosome spontaneously achieves a transient, “unlocked” state is closely correlated with the rate at which the tRNA-like, domain IV/V element of EF-G engages the A site. Following these structural transitions, translocation occurs comparatively fast, suggesting that conformational processes intrinsic to the ribosome determine the rate of translocation. Experiments performed in the presence of non-hydrolyzable GTP analogues and specific antibiotics further reveal that allosterically linked conformational events in EF-G and the ribosome mediate rapid, directional substrate movement and EF-G release. PMID:21057527

  11. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  12. Functional Role of Ribosomal Signatures

    PubMed Central

    Chen, Ke; Eargle, John; Sarkar, Krishnarjun; Gruebele, Martin; Luthey-Schulten, Zaida

    2010-01-01

    Although structure and sequence signatures in ribosomal RNA and proteins are defining characteristics of the three domains of life and instrumental in constructing the modern phylogeny, little is known about their functional roles in the ribosome. In this work, the largest coevolving RNA/protein signatures in the bacterial 30S ribosome are investigated both experimentally and computationally through all-atom molecular-dynamics simulations. The complex includes the N-terminal fragment of the ribosomal protein S4, which is a primary binding protein that initiates 30S small subunit assembly from the 5′ domain, and helix 16 (h16), which is part of the five-way junction in 16S rRNA. Our results show that the S4 N-terminus signature is intrinsically disordered in solution, whereas h16 is relatively stable by itself. The dynamic disordered property of the protein is exploited to couple the folding and binding process to the five-way junction, and the results provide insight into the mechanism for the early and fast binding of S4 in the assembly of the ribosomal small subunit. PMID:21156135

  13. Ral-GTPases: approaching their 15 minutes of fame.

    PubMed

    Feig, Larry A

    2003-08-01

    Andy Warhol, the famous pop artist, once claimed that "in the future everyone will be famous for 15 minutes". The same, it seems, can be said of proteins, because at any given time some proteins become more "fashionable" to study than others. But most proteins have been highly conserved throughout millions of years of evolution, which implies that they all have essential roles in cell biology. Thus, each one will no doubt enter the limelight if the right experiment in the right cell type is done. A good example of this is the Ras-like GTPases (Ral-GTPases), which until recently existed in the shadow of their close cousins--the Ras proto-oncogenes. Recent studies have yielded insights into previously unappreciated roles for Ral-GTPases in intensively investigated disciplines such as vesicle trafficking, cell morphology, transcription and possibly even human oncogenesis. PMID:12888294

  14. Structural Basis for Translation Termination on the 70S Ribosome

    SciTech Connect

    Laurberg, M.; Asahara, H.; Korostelev, A.; Zhu, J.; Trakhanov, S.; Noller, H.F.

    2009-05-20

    At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 {angstrom} resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.

  15. Ribosomal RNA analysis in the diagnosis of Diamond-Blackfan Anaemia.

    PubMed

    Quarello, Paola; Garelli, Emanuela; Carando, Adriana; Mancini, Cecilia; Foglia, Luiselda; Botto, Carlotta; Farruggia, Piero; De Keersmaecker, Kim; Aspesi, Anna; Ellis, Steve R; Dianzani, Irma; Ramenghi, Ugo

    2016-03-01

    Diamond-Blackfan anaemia (DBA) is an inherited disease characterized by pure erythroid aplasia that has been tagged as a 'ribosomopathy'. We report a multi-centre study focused on the analysis of rRNA processing of 53 Italian DBA patients using capillary electrophoresis analysis of rRNA maturation of the 40S and 60S ribosomal subunits. The ratio of 28S/18S rRNA was higher in patients with mutated ribosomal proteins (RPs) of the small ribosomal subunit. In contrast, patients with mutated RPs of the large ribosomal subunit (RPLs) had a lower 28S/18S ratio. The assay reported here would be amenable for development as a diagnostic tool. PMID:26763766

  16. Profiling of Mycoplasma gallisepticum Ribosomes

    PubMed Central

    Fisunov, G. Y.; Evsyutina, D. V.; Arzamasov, A. A.; Butenko, I. O.; Govorun, V. M.

    2015-01-01

    The development of high-throughput technologies is increasingly resulting in identification of numerous cases of low correlation between mRNA and the protein level in cells. These controversial observations were made on various bacteria, such as E. coli, Desulfovibrio vulgaris, and Lactococcus lactis. Thus, it is important to develop technologies, including high-throughput techniques, aimed at studying gene expression regulation at the level of translation. In the current study, we performed proteomic profiling of M. gallisepticum ribosomes and identified high abundant noncanonical proteins. We found that binding of mRNAs to ribosomes is mainly determined by two parameters: (1) abundance of mRNA itself and (2) complimentary interactions between the 3’ end of 16S rRNA and the ribosome binding site in the 5’-untranslated region of mRNA. PMID:26798497

  17. Profiling of Mycoplasma gallisepticum Ribosomes.

    PubMed

    Fisunov, G Y; Evsyutina, D V; Arzamasov, A A; Butenko, I O; Govorun, V M

    2015-01-01

    The development of high-throughput technologies is increasingly resulting in identification of numerous cases of low correlation between mRNA and the protein level in cells. These controversial observations were made on various bacteria, such as E. coli, Desulfovibrio vulgaris, and Lactococcus lactis. Thus, it is important to develop technologies, including high-throughput techniques, aimed at studying gene expression regulation at the level of translation. In the current study, we performed proteomic profiling of M. gallisepticum ribosomes and identified high abundant noncanonical proteins. We found that binding of mRNAs to ribosomes is mainly determined by two parameters: (1) abundance of mRNA itself and (2) complimentary interactions between the 3' end of 16S rRNA and the ribosome binding site in the 5'-untranslated region of mRNA. PMID:26798497

  18. Comprehensive Molecular Structure of the Eukaryotic Ribosome

    PubMed Central

    Taylor, Derek J.; Devkota, Batsal; Huang, Andrew D.; Topf, Maya; Narayanan, Eswar; Sali, Andrej; Harvey, Stephen C.; Frank, Joachim

    2009-01-01

    Despite the emergence of a large number of X-ray crystallographic models of the bacterial 70S ribosome over the past decade, an accurate atomic model of the eukaryotic 80S ribosome is still not available. Eukaryotic ribosomes possess more ribosomal proteins and ribosomal RNA than bacterial ribosomes, which are implicated in extra-ribosomal functions in the eukaryotic cells. By combining cryo-EM with RNA and protein homology modeling, we obtained an atomic model of the yeast 80S ribosome complete with all ribosomal RNA expansion segments and all ribosomal proteins for which a structural homolog can be identified. Mutation or deletion of 80S ribosomal proteins can abrogate maturation of the ribosome, leading to several human diseases. We have localized one such protein unique to eukaryotes, rpS19e, whose mutations are associated with Diamond-Blackfan anemia in humans. Additionally, we characterize crucial and novel interactions between the dynamic stalk base of the ribosome with eukaryotic elongation factor 2. PMID:20004163

  19. New ribosomes for new memories?

    PubMed Central

    Hernández, A Iván; Alarcon, Juan M; Allen, Kim D

    2015-01-01

    Widely thought to be a housekeeping process, the regulation and synthesis of rRNA emerges as a potentially central mechanism for the maintenance of synaptic plasticity and memory. We have recently shown that an essential component of late-phase synaptic plasticity is rRNA biosynthesis — the rate-limiting step in the production of new ribosomes. We hypothesize that a particular population of ribosomes is generated upon learning-associated neural activity to alter the rate of synthesis of plasticity factors at tagged synapses that will support the maintenance of synaptic plasticity and memory. PMID:26479998

  20. EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

    PubMed Central

    Liu, Wei; Chen, Chunlai; Kavaliauskas, Darius; Knudsen, Charlotte R.; Goldman, Yale E.; Cooperman, Barry S.

    2015-01-01

    The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change. PMID:26338772

  1. Chromatographic Purification of Highly Active Yeast Ribosomes

    PubMed Central

    Meskauskas, Arturas; Leshin, Jonathan A.; Dinman, Jonathan D.

    2011-01-01

    Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes. PMID:22042245

  2. Insights into RNA binding by the anticancer drug cisplatin from the crystal structure of cisplatin-modified ribosome

    PubMed Central

    Melnikov, Sergey V.; Söll, Dieter; Steitz, Thomas A.; Polikanov, Yury S.

    2016-01-01

    Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin–RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome—the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity. PMID:27079977

  3. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  4. Studies on Pea Ribosomal Proteins

    PubMed Central

    Lin, Chu-Yung; Chia, Subrina Li-Li; Travis, Robert L.; Key, Joe L.

    1975-01-01

    Ribosomal subunits prepared by NH4Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L2 and L9 as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH4Cl retained L2 and L9, but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl2) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH4Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L2 and L9, showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH4Cl (this apparently leads to a preferential detachment of L2 and L9 at 0.5 m NH4Cl) and that the activity of the purified subunits depends not only on the presence of L2 and L9 but also on the organization of these proteins within the 60S subunits. Images PMID:16659254

  5. Transglutaminase 2 Regulates the GTPase-activating Activity of Bcr*

    PubMed Central

    Yi, Sun-Ju; Groffen, John; Heisterkamp, Nora

    2009-01-01

    Transglutaminase 2 (TG2) is a multifunctional protein that has been implicated in numerous pathologies including that of neurodegeneration and celiac disease, but the molecular interactions that mediate its diverse activities are largely unknown. Bcr and the closely related Abr negatively regulate the small G-protein Rac: loss of their combined function in vivo results in increased reactivity of innate immune cells. Bcr and Abr are GTPase-activating proteins that catalyze the hydrolysis of the GTP bound to Rac. However, how the Bcr and Abr GTPase-activating activity is regulated is not precisely understood. We here report a novel mechanism of regulation through direct protein-protein interaction with TG2. TG2 bound to the Rac-binding pocket in the GTPase-activating domains of Bcr and Abr, blocked Bcr activity and, through this mechanism, increased levels of active GTP-bound Rac and EGF-stimulated membrane ruffling. TG2 exists in at least two different conformations. Interestingly, experiments using TG2 mutants showed that Bcr exhibits preferential binding to the non-compacted conformation of TG2, in which its catalytic domain is exposed, but transamidation is not needed for the interaction. Thus, TG2 regulates levels of cellular GTP-bound Rac and actin cytoskeletal reorganization through a new mechanism involving direct inhibition of Bcr GTPase-activating activity. PMID:19840940

  6. The interdependence of the Rho GTPases and apicobasal cell polarity

    PubMed Central

    Mack, Natalie Ann; Georgiou, Marios

    2014-01-01

    Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease. PMID:25469537

  7. G domain dimerization controls dynamin's assembly-stimulated GTPase activity

    SciTech Connect

    Chappie, Joshua S.; Acharya, Sharmistha; Leonard, Marilyn; Schmid, Sandra L.; Dyda, Fred

    2010-06-14

    Dynamin is an atypical GTPase that catalyses membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin's basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0 {angstrom} resolution crystal structure of a human dynamin 1-derived minimal GTPase-GED fusion protein, which was dimeric in the presence of the transition state mimic GDP.AlF{sub 4}{sup -}. The structure reveals dynamin's catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GTPase-GED fusion protein dimer provides insight into the mechanisms underlying dynamin-catalysed membrane fission.

  8. Structural Mechanisms and Drug Discovery Prospects of Rho GTPases

    PubMed Central

    Smithers, Cameron C.; Overduin, Michael

    2016-01-01

    Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies. PMID:27304967

  9. RhoGAPs and Rho GTPases in platelets.

    PubMed

    Elvers, Margitta

    2016-08-01

    Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets responsible for the formation of filopodia and lamellipodia to strongly increase the platelet surface upon activation. They are involved in platelet activation and aggregate formation including platelet secretion, integrin activation and arterial thrombus formation. The activity of Rho GTPases is tightly controlled by different proteins such as GTPase-activating proteins (GAPs). GAPs stimulate GTP hydrolysis to terminate Rho signaling. The role and impact of GAPs in platelets is not well-defined and many of the RhoGAPs identified are not known to be present in platelets or to have any function in platelets. The recently identified RhoGAPs Oligophrenin1 (OPHN1) and Nadrin regulate the activity of RhoA, Rac1 and Cdc42 and subsequent platelet cytoskeletal reorganization, platelet activation and thrombus formation. In the last years, the analysis of genetically modified mice helped to gain the understanding of Rho GTPases and their regulators in cytoskeletal rearrangements and other Rho mediated cellular processes in platelets. PMID:25639730

  10. Control of T lymphocyte morphology by the GTPase Rho

    NASA Technical Reports Server (NTRS)

    Woodside, Darren G.; Wooten, David K.; Teague, T. Kent; Miyamoto, Yuko J.; Caudell, Eva G.; Udagawa, Taturo; Andruss, Bernard F.; McIntyre, Bradley W.

    2003-01-01

    BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

  11. AMPylation of Rho GTPases Subverts Multiple Host Signaling Processes*

    PubMed Central

    Woolery, Andrew R.; Yu, Xiaobo; LaBaer, Joshua; Orth, Kim

    2014-01-01

    Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules. Herein, we demonstrate that AMPylation of Rho GTPases by VopS is a multifaceted virulence mechanism that counters several host immunity strategies. Activation of NFκB, Erk, and JNK kinase signaling pathways were inhibited in a VopS-dependent manner during infection with Vibrio parahaemolyticus. Phosphorylation and degradation of IKBα were inhibited in the presence of VopS as was nuclear translocation of the NFκB subunit p65. AMPylation also prevented the generation of superoxide by the phagocytic NADPH oxidase complex, potentially by inhibiting the interaction of Rac and p67. Furthermore, the interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a nucleic acid programmable protein array and discovered that Rac1 binds to the protein C1QA, a protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted by AMPylation. We conclude that AMPylation of Rho Family GTPases by VopS results in diverse inhibitory consequences during infection beyond the most obvious phenotype, the collapse of the actin cytoskeleton. PMID:25301945

  12. The interdependence of the Rho GTPases and apicobasal cell polarity.

    PubMed

    Mack, Natalie Ann; Georgiou, Marios

    2014-01-01

    Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease. PMID:25469537

  13. Epithelial junctions and Rho family GTPases: the zonular signalosome

    PubMed Central

    Citi, Sandra; Guerrera, Diego; Spadaro, Domenica; Shah, Jimit

    2014-01-01

    The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of “zonular signalosome” is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors. PMID:25483301

  14. Pattern formation of Rho GTPases in single cell wound healing

    PubMed Central

    Simon, Cory M.; Vaughan, Emily M.; Bement, William M.; Edelstein-Keshet, Leah

    2013-01-01

    The Rho GTPases—Rho, Rac, and Cdc42—control an enormous variety of processes, many of which reflect activation of these GTPases in spatially confined and mutually exclusive zones. By using mathematical models and experimental results to establish model parameters, we analyze the formation and segregation of Rho and Cdc42 zones during Xenopus oocyte wound repair and the role played by Abr, a dual guanine nucleotide exchange factor–GTPase-activating protein, in this process. The Rho and Cdc42 zones are found to be best represented as manifestations of spatially modulated bistability, and local positive feedback between Abr and Rho can account for the maintenance and dynamic properties of the Rho zone. In contrast, the invocation of an Abr-independent positive feedback loop is required to account for Cdc42 spatial bistability. In addition, the model replicates the results of previous in vivo experiments in which Abr activity is manipulated. Further, simulating the model with two closely spaced wounds made nonintuitive predictions about the Rho and Cdc42 patterns; these predictions were confirmed by experiment. We conclude that the model is a useful tool for analysis of Rho GTPase signaling and that the Rho GTPases can be fruitfully considered as components of intracellular pattern formation systems. PMID:23264464

  15. MIRO GTPases in Mitochondrial Transport, Homeostasis and Pathology

    PubMed Central

    Tang, Bor Luen

    2015-01-01

    The evolutionarily-conserved mitochondrial Rho (MIRO) small GTPase is a Ras superfamily member with three unique features. It has two GTPase domains instead of the one found in other small GTPases, and it also has two EF hand calcium binding domains, which allow Ca2+-dependent modulation of its activity and functions. Importantly, it is specifically associated with the mitochondria and via a hydrophobic transmembrane domain, rather than a lipid-based anchor more commonly found in other small GTPases. At the mitochondria, MIRO regulates mitochondrial homeostasis and turnover. In metazoans, MIRO regulates mitochondrial transport and organization at cellular extensions, such as axons, and, in some cases, intercellular transport of the organelle through tunneling nanotubes. Recent findings have revealed a myriad of molecules that are associated with MIRO, particularly the kinesin adaptor Milton/TRAK, mitofusin, PINK1 and Parkin, as well as the endoplasmic reticulum-mitochondria encounter structure (ERMES) complex. The mechanistic aspects of the roles of MIRO and its interactors in mitochondrial homeostasis and transport are gradually being revealed. On the other hand, MIRO is also increasingly associated with neurodegenerative diseases that have roots in mitochondrial dysfunction. In this review, I discuss what is currently known about the cellular physiology and pathophysiology of MIRO functions. PMID:26729171

  16. Structural Mechanisms and Drug Discovery Prospects of Rho GTPases.

    PubMed

    Smithers, Cameron C; Overduin, Michael

    2016-01-01

    Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies. PMID:27304967

  17. Osteoblast differentiation and migration are regulated by dynamin GTPase activity.

    PubMed

    Eleniste, Pierre P; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W; Bruzzaniti, Angela

    2014-01-01

    Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0-21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation. PMID:24387844

  18. Osteoblast differentiation and migration are regulated by Dynamin GTPase activity

    PubMed Central

    Eleniste, Pierre P.; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W.; Bruzzaniti, Angela

    2013-01-01

    Bone formation is controlled by osteoblasts but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0–21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation. PMID:24387844

  19. Characterizing inactive ribosomes in translational profiling.

    PubMed

    Liu, Botao; Qian, Shu-Bing

    2016-01-01

    The broad impact of translational regulation has emerged explosively in the last few years in part due to the technological advance in genome-wide interrogation of gene expression. During mRNA translation, the majority of actively translating ribosomes exist as polysomes in cells with multiple ribosomes loaded on a single transcript. The importance of the monosome, however, has been less appreciated in translational profiling analysis. Here we report that the monosome fraction isolated by sucrose sedimentation contains a large quantity of inactive ribosomes that do not engage on mRNAs to direct translation. We found that the elongation factor eEF2, but not eEF1A, stably resides in these non-translating ribosomes. This unique feature permits direct evaluation of ribosome status under various stress conditions and in the presence of translation inhibitors. Ribosome profiling reveals that the monosome has a similar but not identical pattern of ribosome footprints compared to the polysome. We show that the association of free ribosomal subunits minimally contributes to ribosome occupancy outside of the coding region. Our results not only offer a quantitative method to monitor ribosome availability, but also uncover additional layers of ribosome status needed to be considered in translational profiling analysis. PMID:27335722

  20. Intersubunit movement is required for ribosomal translocation

    PubMed Central

    Horan, Lucas H.; Noller, Harry F.

    2007-01-01

    Translocation of tRNA and mRNA during protein synthesis is believed to be coupled to structural changes in the ribosome. The “ratchet model,” based on cryo-EM reconstructions of ribosome complexes, invokes relative movement of the 30S and 50S ribosomal subunits in this process; however, evidence that directly demonstrates a requirement for intersubunit movement during translocation is lacking. To address this problem, we created an intersubunit disulfide cross-link to restrict potential movement. The cross-linked ribosomes were unable to carry out polypeptide synthesis; this inhibition was completely reversed upon reduction of the disulfide bridge. In vitro assays showed that the cross-linked ribosomes were specifically blocked in elongation factor G-dependent translocation. These findings show that intersubunit movement is required for ribosomal translocation, accounting for the universal two-subunit architecture of ribosomes. PMID:17360328

  1. Alcoholic Liver Disease and the Mitochondrial Ribosome

    PubMed Central

    Cahill, Alan; Sykora, Peter

    2009-01-01

    Summary Chronic alcohol consumption has been shown to severely compromise mitochondrial protein synthesis. Hepatic mitochondria isolated from alcoholic animals contain decreased levels of respiratory complexes and display depressed respiration rates when compared to pair-fed controls. One underlying mechanism for this involves ethanol-elicited alterations in the structural and functional integrity of the mitochondrial ribosome. Ethanol feeding results in ribosomal changes that include decreased sedimentation rates, larger hydrodynamic volumes, increased levels of unassociated subunits and changes in the levels of specific ribosomal proteins. The methods presented in this chapter detail how to isolate mitochondrial ribosomes, determine ribosomal activity, separate ribosomes into nucleic acid and protein, and perform two-dimensional nonequilibrium pH gradient electrophoretic polyacrylamide gel electrophoresis to separate and subsequently identify mitochondrial ribosomal proteins. PMID:18369931

  2. [About the ribosomal biogenesis in human].

    PubMed

    Tafforeau, Lionel

    2015-01-01

    Ribosomes are cellular ribonucleoprotein particles required for a fundamental mechanism, translation of the genetic information into proteins. Ribosome biogenesis is a highly complex pathway involving many maturation steps: ribosomal RNA (rRNA) synthesis, rRNA processing, pre-rRNA modifications, its assembly with ribosomal proteins in the nuceolus, export of the subunit precursors to the nucleoplasm and the cytoplasm. Ribosome biogenesis has mainly being investigated in yeast during these last 25 years. However, recent works have shown that, despite many similarities between yeast and human ribosome structure and biogenesis, human pre-rRNA processing is far more complex than in yeast. In order to better understand diseases related to a malfunction in ribosome synthesis, the ribosomopathies, research should be conducted directly in human cells and animal models. PMID:26152166

  3. The dynamics of spatio-temporal Rho GTPase signaling: formation of signaling patterns

    PubMed Central

    Fritz, Rafael Dominik; Pertz, Olivier

    2016-01-01

    Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity. PMID:27158467

  4. The function of RhoGTPases in axon ensheathment and myelination

    PubMed Central

    Feltri, M. Laura; Suter, Ueli; Relvas, João B.

    2008-01-01

    RhoGTPases are molecular switches that integrate extracellular signals to perform diverse cellular responses. This ability relies on the network of proteins regulating RhoGTPases activity and localization, and on the interaction of RhoGTPases with many different cellular effectors. Myelination is an ideal place for RhoGTPases regulation, as it is the result of fine orchestration of many stimuli from at least two cell types. Recent work has revealed that RhoGTPases are required for Schwann cells to sort, ensheath and myelinate axons. Here we will review recent advances showing the critical roles for RhoGTPases in various aspects of Schwann development and myelination, including the recent discovery of their involvement in Charcot-Marie-Tooth disease. Comparison with potential roles of RhoGTPases in central nervous system myelination will be drawn. PMID:18803320

  5. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

  6. Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

    PubMed Central

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

  7. Neutron scattering in the ribosome structure

    NASA Astrophysics Data System (ADS)

    Serdyuk, Igor N.

    1997-02-01

    Thermal neutron scattering has become a powerful instrument for studying the ribosome and its components. The application of neutron scattering allowed to establish some principal features of the ribosome structure: non-homogeneous distribution of the RNA and protein within ribosomal particles, the RNA role as a framework in the arrangement and maintenance of the structure of ribosomal particles, and the globular character of ribosomal proteins. The use of selective deuteration of separate ribosomal proteins in combination with the triangulation method revealed mutual spatial arrangement (the 3D-map) of all the ribosomal proteins within the small particle and in the most part of the large ribosomal particle. An essential impact has been made in the structural studies of ribosomes with the development of novel experimental approaches: triple isotopic substitution and spin contrast variation. These approaches with direct interpretation of spherical harmonics provide new possibilities for constructing models of ribosomal particles, opening principally new perspectives for joint use of X-ray synchrotron diffraction in crystals and small-angle neutron scattering in solution.

  8. Immune control of phagosomal bacteria by p47 GTPases.

    PubMed

    MacMicking, John D

    2005-02-01

    Sequestered from the action of complement, antibody and lytic peptides, phagosomal pathogens pose a unique problem for the innate immune system both in terms of detection and disposal. An immunologically induced 47-kDa (p47) GTPase family recruited to nascent phagosomes (PGs) has provided new insights into how vertebrates deal with facultative bacteria occupying a vacuolar niche. Research over the past 2 years in particular has identified several molecular determinants that underlie the membrane trafficking functions of LRG-47 and other p47 GTPases as part of a PG remodeling program. When coupled to signals issuing from pathogen-specific Toll-like receptors, the p47 proteins may constitute a novel sensory system enlisted by mammals, birds and fish to decode the language of immune recognition against this particular class of infectious agents. PMID:15694860

  9. Regulation of autophagy by the Rab GTPase network

    PubMed Central

    Ao, X; Zou, L; Wu, Y

    2014-01-01

    Autophagy (macroautophagy) is a highly conserved intracellular and lysosome-dependent degradation process in which autophagic substrates are enclosed and degraded by a double-membrane vesicular structure in a continuous and dynamic vesicle transport process. The Rab protein is a small GTPase that belongs to the Ras-like GTPase superfamily and regulates the vesicle traffic process. Numerous Rab proteins have been shown to be involved in various stages of autophagy. Rab1, Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B participate in autophagosome formation, whereas Rab9 is required in non-canonical autophagy. Rab7, Rab8B, and Rab24 have a key role in autophagosome maturation. Rab8A and Rab25 are also involved in autophagy, but their role is unknown. Here, we summarize new findings regarding the involvement of Rabs in autophagy and provide insights regarding future research on the mechanisms of autophagy regulation. PMID:24440914

  10. Modelling Rho GTPase biochemistry to predict collective cell migration

    NASA Astrophysics Data System (ADS)

    Merchant, Brian; Feng, James

    The collective migration of cells, due to individual cell polarization and intercellular contact inhibition of locomotion, features prominently in embryogenesis and metastatic cancers. Existing methods for modelling collectively migrating cells tend to rely either on highly abstracted agent-based models, or on continuum approximations of the group. Both of these frameworks represent intercellular interactions such as contact inhibition of locomotion as hard-coded rules defining model cells. In contrast, we present a vertex-dynamics framework which predicts polarization and contact inhibition of locomotion naturally from an underlying model of Rho GTPase biochemistry and cortical mechanics. We simulate the interaction between many such model cells, and study how modulating Rho GTPases affects migratory characteristics of the group, in the context of long-distance collective migration of neural crest cells during embryogenesis.

  11. Structural Insights Into Ribosome Recycling Factor Interactions with the 70S Ribosome

    PubMed Central

    Pai, Raj D.; Zhang, Wen; Schuwirth, Barbara S.; Hirokawa, Go; Kaji, Hideko; Kaji, Akira; Cate, Jamie H.D.

    2009-01-01

    SUMMARY At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with Elongation Factor G (EF-G) to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center (PTC). Upon binding of either E. coli or T. thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix H69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits, termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of H69 involves an ordered to disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between Domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling. PMID:18234219

  12. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32

  13. Emerging nexus between RAB GTPases, autophagy and neurodegeneration.

    PubMed

    Jain, Navodita; Ganesh, Subramaniam

    2016-05-01

    The RAB class of small GTPases includes the major regulators of intracellular communication, which are involved in vesicle generation through fusion and fission, and vesicular trafficking. RAB proteins also play an imperative role in neuronal maintenance and survival. Recent studies in the field of neurodegeneration have also highlighted the process of autophagy as being essential for neuronal maintenance. Here we review the emerging roles of RAB proteins in regulating macroautophagy and its impact in the context of neurodegenerative diseases. PMID:26985808

  14. Ral GTPases regulate exocyst assembly through dual subunit interactions.

    PubMed

    Moskalenko, Serge; Tong, Chao; Rosse, Carine; Mirey, Gladys; Formstecher, Etienne; Daviet, Laurent; Camonis, Jacques; White, Michael A

    2003-12-19

    Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84. PMID:14525976

  15. Targeting Rho-GTPases in immune cell migration and inflammation

    PubMed Central

    Biro, Maté; Munoz, Marcia A; Weninger, Wolfgang

    2014-01-01

    Leukocytes are unmatched migrators capable of traversing barriers and tissues of remarkably varied structural composition. An effective immune response relies on the ability of its constituent cells to infiltrate target sites. Yet, unwarranted mobilization of immune cells can lead to inflammatory diseases and tissue damage ranging in severity from mild to life-threatening. The efficacy and plasticity of leukocyte migration is driven by the precise spatiotemporal regulation of the actin cytoskeleton. The small GTPases of the Rho family (Rho-GTPases), and their immediate downstream effector kinases, are key regulators of cellular actomyosin dynamics and are therefore considered prime pharmacological targets for stemming leukocyte motility in inflammatory disorders. This review describes advances in the development of small-molecule inhibitors aimed at modulating the Rho-GTPase-centric regulatory pathways governing motility, many of which stem from studies of cancer invasiveness. These inhibitors promise the advent of novel treatment options with high selectivity and potency against immune-mediated pathologies. Linked Articles This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24 PMID:24571448

  16. Interaction of LRRK2 with kinase and GTPase signaling cascades

    PubMed Central

    Boon, Joon Y.; Dusonchet, Julien; Trengrove, Chelsea; Wolozin, Benjamin

    2014-01-01

    LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD. PMID:25071441

  17. Modulation of Rab GTPase function by a protein phosphocholine transferase.

    PubMed

    Mukherjee, Shaeri; Liu, Xiaoyun; Arasaki, Kohei; McDonough, Justin; Galán, Jorge E; Roy, Craig R

    2011-09-01

    The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions. PMID:21822290

  18. Control of ribosome formation in rat heart

    SciTech Connect

    Russo, L.A.

    1987-01-01

    Diabetes of 9 days duration produced a 17% diminution in the rate of total protein synthesis in rat hearts perfused as Langendorff preparations supplied with glucose, plasma levels of amino acids, and 400 ..mu..U/ml insulin. This reduction was attributable to a decrease in efficiency of protein synthesis and total RNA content. Total messenger RNA content decreased in diabetic hearts in proportion to the reduction in total RNA. Diabetes also resulted in diminished ribosome content as reflected by the induction in total RNA. Ribosome production was investigated by monitoring incorporation of (/sup 3/H)phenylalanine into the proteins of cytoplasmic ribosomes. Rates of ribosome formation in diabetic hearts were as fast as control rates in the presence of insulin, and were faster than control rates in the absence of the hormone. These results indicated that ribosome content fell in diabetic hearts despite unchanged or faster rates of ribosome formation.

  19. Seeing is Believing in Ribosome Assembly.

    PubMed

    Warner, Jonathan R

    2016-07-14

    Many proteins have been implicated genetically and biochemically in the assembly of eukaryotic ribosomes. Now, Kornprobst et al. show us how they are put together with a cryoEM structure of the 90S processome that initiates ribosome assembly, revealing the arrangement of U3 RNA and the several UTP complexes that form a chaperone-like structure around and within the developing 40S ribosomal subunit. PMID:27419867

  20. Tricks an IRES uses to enslave ribosomes

    PubMed Central

    2012-01-01

    In eukaryotes, mRNAs are primarily translated through a cap-dependent mechanism whereby initiation factors recruit the 40S ribosomal subunit to a cap structure at the 5’ end of the mRNA. However, some viral and cellular messages initiate protein synthesis without a cap. They use a structured RNA element termed an internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit. IRESs were discovered over 20 years ago but only recently have studies using a model IRES from dicistroviruses expanded our understanding of how a three dimensional RNA structure can capture and manipulate the ribosome to initiate translation. PMID:22944245

  1. Scattering studies on ribosomes in solution

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, V.

    1986-02-01

    Ribosomes are organelles that play a central role in protein synthesis. They are complexes of protein and nucleic acid, and can be analysed as two-component systems by neutron scattering. Moreover, ribosomes can be biochemically prepared that have specific proteins deuterated. Both these properties have been exploited to study the structure of the ribosome by neutron scattering. This article reviews the studies carried out on the small ribosomal subunit, and describes a recent study that has resolved a conflict between the results of two classes of experiments.

  2. Ribosome biogenesis in the yeast Saccharomyces cerevisiae.

    PubMed

    Woolford, John L; Baserga, Susan J

    2013-11-01

    Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. PMID:24190922

  3. Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

    PubMed Central

    Catz, Sergio Daniel

    2013-01-01

    The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

  4. Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection

    PubMed Central

    Hilbi, Hubert; Rothmeier, Eva; Hoffmann, Christine; Harrison, Christopher F

    2014-01-01

    Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires’ disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cell's endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells. PMID:25496424

  5. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  6. Ribosome Mechanics Informs about Mechanism.

    PubMed

    Zimmermann, Michael T; Jia, Kejue; Jernigan, Robert L

    2016-02-27

    The essential aspects of the ribosome's mechanism can be extracted from coarse-grained simulations, including the ratchet motion, the movement together of critical bases at the decoding center, and movements of the peptide tunnel lining that assist in the expulsion of the synthesized peptide. Because of its large size, coarse graining helps to simplify and to aid in the understanding of its mechanism. Results presented here utilize coarse-grained elastic network modeling to extract the dynamics, and both RNAs and proteins are coarse grained. We review our previous results, showing the well-known ratchet motions and the motions in the peptide tunnel and in the mRNA tunnel. The motions of the lining of the peptide tunnel appear to assist in the expulsion of the growing peptide chain, and clamps at the ends of the mRNA tunnel with three proteins ensure that the mRNA is held tightly during decoding and essential for the helicase activity at the entrance. The entry clamp may also assist in base recognition to ensure proper selection of the incoming tRNA. The overall precision of the ribosome machine-like motions is remarkable. PMID:26687034

  7. Mitomycin C Inhibits Ribosomal RNA

    PubMed Central

    Snodgrass, Ryan G.; Collier, Abby C.; Coon, Amy E.; Pritsos, Chris A.

    2010-01-01

    Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds. PMID:20418373

  8. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration

    PubMed Central

    Stankiewicz, Trisha R.; Linseman, Daniel A.

    2014-01-01

    The Rho family of GTPases belongs to the Ras superfamily of low molecular weight (∼21 kDa) guanine nucleotide binding proteins. The most extensively studied members are RhoA, Rac1, and Cdc42. In the last few decades, studies have demonstrated that Rho family GTPases are important regulatory molecules that link surface receptors to the organization of the actin and microtubule cytoskeletons. Indeed, Rho GTPases mediate many diverse critical cellular processes, such as gene transcription, cell–cell adhesion, and cell cycle progression. However, Rho GTPases also play an essential role in regulating neuronal morphology. In particular, Rho GTPases regulate dendritic arborization, spine morphogenesis, growth cone development, and axon guidance. In addition, more recent efforts have underscored an important function for Rho GTPases in regulating neuronal survival and death. Interestingly, Rho GTPases can exert either a pro-survival or pro-death signal in neurons depending upon both the cell type and neurotoxic insult involved. This review summarizes key findings delineating the involvement of Rho GTPases and their effectors in the regulation of neuronal survival and death. Collectively, these results suggest that dysregulation of Rho family GTPases may potentially underscore the etiology of some forms of neurodegenerative disease such as amyotrophic lateral sclerosis. PMID:25339865

  9. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration.

    PubMed

    Stankiewicz, Trisha R; Linseman, Daniel A

    2014-01-01

    The Rho family of GTPases belongs to the Ras superfamily of low molecular weight (∼21 kDa) guanine nucleotide binding proteins. The most extensively studied members are RhoA, Rac1, and Cdc42. In the last few decades, studies have demonstrated that Rho family GTPases are important regulatory molecules that link surface receptors to the organization of the actin and microtubule cytoskeletons. Indeed, Rho GTPases mediate many diverse critical cellular processes, such as gene transcription, cell-cell adhesion, and cell cycle progression. However, Rho GTPases also play an essential role in regulating neuronal morphology. In particular, Rho GTPases regulate dendritic arborization, spine morphogenesis, growth cone development, and axon guidance. In addition, more recent efforts have underscored an important function for Rho GTPases in regulating neuronal survival and death. Interestingly, Rho GTPases can exert either a pro-survival or pro-death signal in neurons depending upon both the cell type and neurotoxic insult involved. This review summarizes key findings delineating the involvement of Rho GTPases and their effectors in the regulation of neuronal survival and death. Collectively, these results suggest that dysregulation of Rho family GTPases may potentially underscore the etiology of some forms of neurodegenerative disease such as amyotrophic lateral sclerosis. PMID:25339865

  10. Established and emerging fluorescence-based assays for G-protein function: Ras-superfamily GTPases.

    PubMed

    Rojas, Rafael J; Kimple, Randall J; Rossman, Kent L; Siderovski, David P; Sondek, John

    2003-06-01

    Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases. PMID:12769685

  11. Science Learning Centres Roundup

    ERIC Educational Resources Information Center

    Education in Science, 2010

    2010-01-01

    The national network of Science Learning Centres aims to raise the quality of science teaching from Key Stage 1 through post-16 (ages 5-19). Short courses are provided locally through the regional Science Learning Centres and longer, more intensive programmes are available at the National Science Learning Centre in York. There are a growing number…

  12. Ribosome hijacking: a role for small protein B during trans-translation

    PubMed Central

    Nonin-Lecomte, Sylvie; Germain-Amiot, Noella; Gillet, Reynald; Hallier, Marc; Ponchon, Luc; Dardel, Frédéric; Felden, Brice

    2009-01-01

    Tight recognition of codon–anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the ‘tmRNA–SmpB' system (transfer messenger RNA–small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon–anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon–anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for ΔsmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon. PMID:19132006

  13. Biochemical characterization of three mycobacterial ribosomal fractions.

    PubMed

    Portelance, V; Beaudet, R

    1983-02-01

    The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity. PMID:6189570

  14. Ribosome flow model with positive feedback

    PubMed Central

    Margaliot, Michael; Tuller, Tamir

    2013-01-01

    Eukaryotic mRNAs usually form a circular structure; thus, ribosomes that terminatae translation at the 3′ end can diffuse with increased probability to the 5′ end of the transcript, initiating another cycle of translation. This phenomenon describes ribosomal flow with positive feedback—an increase in the flow of ribosomes terminating translating the open reading frame increases the ribosomal initiation rate. The aim of this paper is to model and rigorously analyse translation with feedback. We suggest a modified version of the ribosome flow model, called the ribosome flow model with input and output. In this model, the input is the initiation rate and the output is the translation rate. We analyse this model after closing the loop with a positive linear feedback. We show that the closed-loop system admits a unique globally asymptotically stable equilibrium point. From a biophysical point of view, this means that there exists a unique steady state of ribosome distributions along the mRNA, and thus a unique steady-state translation rate. The solution from any initial distribution will converge to this steady state. The steady-state distribution demonstrates a decrease in ribosome density along the coding sequence. For the case of constant elongation rates, we obtain expressions relating the model parameters to the equilibrium point. These results may perhaps be used to re-engineer the biological system in order to obtain a desired translation rate. PMID:23720534

  15. Evolution of the ribosome at atomic resolution

    PubMed Central

    Petrov, Anton S.; Bernier, Chad R.; Hsiao, Chiaolong; Norris, Ashlyn M.; Kovacs, Nicholas A.; Waterbury, Chris C.; Stepanov, Victor G.; Harvey, Stephen C.; Fox, George E.; Wartell, Roger M.; Hud, Nicholas V.; Williams, Loren Dean

    2014-01-01

    The origins and evolution of the ribosome, 3–4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be “observed” by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call “insertion fingerprints.” Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel. PMID:24982194

  16. Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

    PubMed Central

    Pavlov, Michael Y; Antoun, Ayman; Lovmar, Martin; Ehrenberg, Måns

    2008-01-01

    We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine–Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G. PMID:18497739

  17. Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

    PubMed

    Tereshchenkov, A G; Shishkina, A V; Tashlitsky, V N; Korshunova, G A; Bogdanov, A A; Sumbatyan, N V

    2016-04-01

    Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramphenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3-10 µM, which is 100-fold lower than the corresponding values for chloramphenicol amine-ribosome complex. Interaction of the chloramphenicol peptide analogs with ribosomes was simulated by molecular docking, and the most probable contacts of "stop peptide" motifs with the elements of nascent peptide exit tunnel were identified. PMID:27293096

  18. Differential Stoichiometry among Core Ribosomal Proteins.

    PubMed

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-11-01

    Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  19. Viral IRES RNA structures and ribosome interactions.

    PubMed

    Kieft, Jeffrey S

    2008-06-01

    In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES-ribosome complexes are revealing the structural basis of viral IRES' 'hijacking' of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes. PMID:18468443

  20. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  1. Ribosome defects in disorders of erythropoiesis.

    PubMed

    Narla, Anupama; Hurst, Slater N; Ebert, Benjamin L

    2011-02-01

    Over the past decade, genetic lesions that cause ribosome dysfunction have been identified in both congenital and acquired human disorders. These discoveries have established a new category of disorders, known as ribosomopathies, in which the primary pathophysiology is related to impaired ribosome function. The protoptypical disorders are Diamond-Blackfan anemia, a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome. In both of these disorders, impaired ribosome function causes a severe macrocytic anemia. In this review, we will discuss the evidence that defects in ribosomal biogenesis cause the hematologic phenotype of Diamond-Blackfan anemia and the 5q- syndrome. We will also explore the potential mechanisms by which a ribosomal defect, which would be expected to have widespread consequences, may lead to specific defects in erythropoiesis. PMID:21279816

  2. Viral IRES RNA structures and ribosome interactions

    PubMed Central

    Kieft, Jeffrey S.

    2009-01-01

    In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide ‘cap’ on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES–ribosome complexes are revealing the structural basis of viral IRES’ ‘hijacking’ of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes. PMID:18468443

  3. Isoprenoids, Small GTPases and Alzheimer’s Disease

    PubMed Central

    Hooff, Gero P.; Wood, W. Gibson; Müller, Walter E.; Eckert, Gunter P.

    2010-01-01

    The mevalonate-pathway is a crucial metabolic pathway for most eukaryotic cells. Cholesterol is a highly recognized product of this pathway but growing interest is being given to the synthesis and functions of isoprenoids. Isoprenoids are a complex class of biologically active lipids including for example, dolichol, ubiquinone, farnesylpyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Early work had shown that the long-chain isoprenoid dolichol is decreased, but that dolichyl-phosphate and ubiquinone are elevated in brains of Alzheimer´s diseased (AD) patients. Until recently, levels of their biological active precursors FPP and GGPP were unknown. These short-chain isoprenoids are critical in the post translational modification of certain proteins which function as molecular switches in numerous, signaling pathways. The major protein families belong to the superfamily of small GTPases, consisting of roughly 150 members. Recent experimental evidence indicated that members of the small GTPases are involved in AD pathogenesis and stimulated interest in the role of FPP and GGPP in protein prenylation and cell function. A straightforward prediction derived from those studies was that FPP and GGPP levels would be elevated in AD brains as compared with normal neurological controls. For the first time, recent evidence shows significantly elevated levels of FPP and GGPP in human AD brain tissue. Cholesterol levels did not differ between AD and control samples. One obvious conclusion is that homeostasis of FPP and GGPP but not of cholesterol is specifically targeted in AD. Since prenylation of small GTPases by FPP or GGPP is indispensable for their proper function we are proposing that these two isoprenoids are up-regulated in AD resulting in an over abundance of certain prenylated proteins which contributes to neuronal dysfunction. PMID:20382260

  4. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of active Gα protein. PMID:27124090

  5. Rho GTPase Transcriptome Analysis Reveals Oncogenic Roles for Rho GTPase-Activating Proteins in Basal-like Breast Cancers.

    PubMed

    Lawson, Campbell D; Fan, Cheng; Mitin, Natalia; Baker, Nicole M; George, Samuel D; Graham, David M; Perou, Charles M; Burridge, Keith; Der, Channing J; Rossman, Kent L

    2016-07-01

    The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826-37. ©2016 AACR. PMID:27216196

  6. Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins.

    PubMed

    Jaworski, A J; Wilson, J B

    1989-10-01

    Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates. PMID:2776972

  7. Review: Ras GTPases and myosin: Qualitative conservation and quantitative diversification in signal and energy transduction.

    PubMed

    Mueller, Matthias P; Goody, Roger S

    2016-08-01

    Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016. PMID:27018658

  8. TBC-Domain GAPs for Rab GTPases Accelerate GTP Hydrolysis by a Dual-Finger Mechanism

    SciTech Connect

    Pan,X.; Eathiraj, S.; Lambright, D.

    2006-01-01

    Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain-Rab-GTPase-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.

  9. Ribosomopathies: human disorders of ribosome dysfunction.

    PubMed

    Narla, Anupama; Ebert, Benjamin L

    2010-04-22

    Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases. PMID:20194897

  10. RhoA GTPase inhibition organizes contraction during epithelial morphogenesis.

    PubMed

    Mason, Frank M; Xie, Shicong; Vasquez, Claudia G; Tworoger, Michael; Martin, Adam C

    2016-08-29

    During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding. PMID:27551058

  11. Ras Family Small GTPase-mediated Neuroprotective Signaling in Stroke

    PubMed Central

    Shi, Geng-Xian; Andres, Douglas A.; Cai, Weikang

    2012-01-01

    Selective neuronal cell death is one of the major causes of neuronal damage following stroke, and cerebral cells naturally mobilize diverse survival signaling pathways to protect against ischemia. Importantly, therapeutic strategies designed to improve endogenous anti-apoptotic signaling appear to hold great promise in stroke treatment. While a variety of complex mechanisms have been implicated in the pathogenesis of stroke, the overall mechanisms governing the balance between cell survival and death are not well-defined. Ras family small GTPases are activated following ischemic insults, and in turn, serve as intrinsic switches to regulate neuronal survival and regeneration. Their ability to integrate diverse intracellular signal transduction pathways makes them critical regulators and potential therapeutic targets for neuronal recovery after stroke. This article highlights the contribution of Ras family GTPases to neuroprotective signaling cascades, including mitogen-activated protein kinase (MAPK) family protein kinase- and AKT/PKB-dependent signaling pathways as well as the regulation of cAMP response element binding (CREB), Forkhead box O (FoxO) and hypoxia-inducible factor 1(HIF1) transcription factors, in stroke. PMID:21521171

  12. Cdc42 GTPase dynamics control directional growth responses

    PubMed Central

    Brand, Alexandra C.; Morrison, Emma; Milne, Stephen; Gonia, Sara; Gale, Cheryl A.; Gow, Neil A. R.

    2014-01-01

    Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca2+ influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca2+ influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca2+. Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca2+. Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca2+. The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca2+ influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca2+-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance. PMID:24385582

  13. Novel peptide recognized by RhoA GTPase.

    PubMed

    Drulis-Fajdasz, Dominika; Jelen, Filip; Oleksy, Arkadiusz; Otlewski, Jacek

    2006-01-01

    A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein. PMID:17019437

  14. Regulators and Effectors of Arf GTPases in Neutrophils

    PubMed Central

    Gamara, Jouda; Chouinard, François; Davis, Lynn; Aoudjit, Fawzi; Bourgoin, Sylvain G.

    2015-01-01

    Polymorphonuclear neutrophils (PMNs) are key innate immune cells that represent the first line of defence against infection. They are the first leukocytes to migrate from the blood to injured or infected sites. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of migrating PMNs. These phagocytes actively engulf microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens with bactericidal compounds. Association of the NADPH oxidase complex at the phagosomal membrane for production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. G protein-dependent signalling pathways tightly control PMN functions. In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses. GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells. The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology. PMID:26609537

  15. Dendritic spine geometry can localize GTPase signaling in neurons

    PubMed Central

    Ramirez, Samuel A.; Raghavachari, Sridhar; Lew, Daniel J.

    2015-01-01

    Dendritic spines are the postsynaptic terminals of most excitatory synapses in the mammalian brain. Learning and memory are associated with long-lasting structural remodeling of dendritic spines through an actin-mediated process regulated by the Rho-family GTPases RhoA, Rac, and Cdc42. These GTPases undergo sustained activation after synaptic stimulation, but whereas Rho activity can spread from the stimulated spine, Cdc42 activity remains localized to the stimulated spine. Because Cdc42 itself diffuses rapidly in and out of the spine, the basis for the retention of Cdc42 activity in the stimulated spine long after synaptic stimulation has ceased is unclear. Here we model the spread of Cdc42 activation at dendritic spines by means of reaction-diffusion equations solved on spine-like geometries. Excitable behavior arising from positive feedback in Cdc42 activation leads to spreading waves of Cdc42 activity. However, because of the very narrow neck of the dendritic spine, wave propagation is halted through a phenomenon we term geometrical wave-pinning. We show that this can account for the localization of Cdc42 activity in the stimulated spine, and, of interest, retention is enhanced by high diffusivity of Cdc42. Our findings are broadly applicable to other instances of signaling in extreme geometries, including filopodia and primary cilia. PMID:26337387

  16. Regulation of Neurotrophin-Induced Axonal Responses via Rho GTPases

    PubMed Central

    HANDEÖZDINLER, P.; ERZURUMLU, REHA S.

    2014-01-01

    Nerve growth factor (NGF) and related neurotrophins induce differential axon growth patterns from embryonic sensory neurons. In wholemount explant cultures of embryonic rat trigeminal ganglion and brainstem or in dissociated cell cultures of the trigeminal ganglion, exogenous supply of NGF leads to axonal elongation, whereas neurotrophin-3 (NT-3) treatment leads to short branching and arborization. Axonal responses to neurotrophins might be mediated via the Rho GTPases. To investigate this possibility, we prepared wholemount trigeminal pathway cultures from E15 rats. We infected the ganglia with recombinant vaccinia viruses that express GFP-tagged dominant negative Rac, Rho, or constitutively active Rac or treated the cultures with lysophosphatitic acid (LPA) to activate Rho. We then examined axonal responses to NGF by use of the lipophilic tracer DiI. Rac activity induced longer axonal growth from the central trigeminal tract, whereas the dominant negative construct of Rac eliminated NGF-induced axon outgrowth. Rho activity also significantly reduced, and the Rho dominant negative construct increased, axon growth from the trigeminal tract. Similar alterations in axonal responses to NT-3 and brain-derived neurotrophic factor were also noted. Our results demonstrate that Rho GTPases play a major role in neurotrophin-induced axonal differentiation of embryonic trigeminal axons. PMID:11559894

  17. Ribonucleic acid and ribosomes of Bacillus stearothermophilus.

    PubMed

    Saunders, G F; Campbell, L L

    1966-01-01

    Saunders, Grady F. (University of Illinois, Urbana), and L. Leon Campbell. Ribonucleic acid and ribosomes of Bacillus stearothermophilus. J. Bacteriol. 91:332-339. 1966.-The ability of some thermophilic bacteria to grow at temperatures as high as 76 C emphasizes the remarkable thermal stability of their crucial macromolecules. An investigation of the ribonucleic acid (RNA) and ribosomes of Bacillus stearothermophilus was conducted. Washed log-phase cells were disrupted either by sonic treatment or by alumina grinding in 10(-2)m MgCl(2)-10(-2)m tris-(hydroxymethyl)aminomethane buffer, pH 7.4 (TM buffer). Ultracentrifugal analysis revealed peaks at 72.5S, 101S, and 135S, with the 101S peak being the most prominent. By lowering the Mg(++) concentration to 10(-3)m, the ribosome preparation was dissociated to give 40S, 31S, and 54S peaks. These in turn were reassociated in the presence of 10(-2)m Mg(++) to give the larger 73S and 135S particles. When heated in TM buffer, Escherichia coli ribosomes began a gradual dissociation at 58 C, and at 70 C underwent a large hyperchromic shift with a T(m) at 72.8 C. In contrast, B. stearothermophilus ribosomes did not show a hyperchromic shift below 70 C; they had a T(m) of 77.9 C. The thermal denaturation curves of the 4S, 16S, and 23S RNA from both organisms were virtually identical. The gross amino acid composition of B. stearothermophilus ribosomes showed no marked differences from that reported for E. coli ribosomes. These data suggest that the unusual thermal stability of B. stearothermophilus ribosomes may reflect either an unusual packing arrangement of the protein to the RNA or differences in the primary structure of the ribosomal proteins. PMID:5903099

  18. Ribosome origins: The relative age of 23S rRNA Domains

    NASA Astrophysics Data System (ADS)

    Hury, James; Nagaswamy, Uma; Larios-Sanz, Maia; Fox, George E.

    2006-08-01

    The modern ribosome and its component RNAs are quite large and it is likely that at an earlier time they were much smaller. Hence, not all regions of the modern ribosomal RNAs (rRNA) are likely to be equally old. In the work described here, it is hypothesized that the oldest regions of the RNAs will usually be highly integrated into the machinery. When this is the case, an examination of the interconnectivity between local RNA regions can provide insight to the relative age of the various regions. Herein, we describe an analysis of all known long-range RNA/RNA interactions within the 23S rRNA and between the 23S rRNA and the 16S rRNA in order to assess the interconnectivity between the usual Domains as defined by secondary structure. Domain V, which contains the peptidyl transferase center is centrally located, extensively connected, and therefore likely to be the oldest region. Domain IV and Domain II are extensively interconnected with both themselves and Domain V. A portion of Domain IV is also extensively connected with the 30S subunit and hence Domain IV may be older than Domain II. These results are consistent with other evidence relating to the relative age of RNA regions. Although the relative time of addition of the GTPase center can not be reliably deduced it is pointed out that the development of this may have dramatically affected the progenotes that preceded the last common ancestor.

  19. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  20. Neurolastin, a dynamin family GTPase, regulates excitatory synapses and spine density

    PubMed Central

    Madan Lomash, Richa; Gu, Xinglong; Youle, Richard J.; Lu, Wei; Roche, Katherine W.

    2015-01-01

    SUMMARY Membrane trafficking and spinogenesis contribute significantly to changes in synaptic strength during development and in various paradigms of synaptic plasticity. GTPases of the dynamin family are key players regulating membrane trafficking. Here, we identify a brain-specific dynamin family GTPase, neurolastin (RNF112/Znf179), with closest homology to atlastin. We demonstrate that neurolastin has functional GTPase and RING domains, making it a unique protein identified with this multi-enzymatic domain organization. We also show that neurolastin is a peripheral membrane protein, which localizes to endosomes and affects endosomal membrane dynamics via its RING domain. In addition, neurolastin knockout mice have fewer dendritic spines, and rescue of the wildtype phenotype requires both the GTPase and RING domains. Furthermore, we find fewer functional synapses and reduced paired pulse facilitation in neurolastin knockout mice. Thus, we identify neurolastin as a dynamin family GTPase that affects endosome size and spine density. PMID:26212327

  1. A new system for naming ribosomal proteins

    PubMed Central

    Ban, Nenad; Beckmann, Roland; Cate, Jamie HD; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis LJ; Lindahl, Lasse; Liljas, Anders; Lipton, Jeffrey M; McAlear, Michael A; Moore, Peter B; Noller, Harry F; Ortega, Joaquin; Panse, Vikram Govind; Ramakrishnan, V; Spahn, Christian MT; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

    2015-01-01

    A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as ‘new system’ names. PMID:24524803

  2. The economics of ribosome biosynthesis in yeast.

    PubMed

    Warner, J R

    1999-11-01

    In a rapidly growing yeast cell, 60% of total transcription is devoted to ribosomal RNA, and 50% of RNA polymerase II transcription and 90% of mRNA splicing are devoted to ribosomal proteins (RPs). Coordinate regulation of the approximately 150 rRNA genes and 137 RP genes that make such prodigious use of resources is essential for the economy of the cell. This is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes, leading to major implications for the expression of other genes as well. PMID:10542411

  3. Computational studies of molecular machines: the ribosome.

    PubMed

    Sanbonmatsu, Karissa Y

    2012-04-01

    The past decade has produced an avalanche of experimental data on the structure and dynamics of the ribosome. Groundbreaking studies in structural biology and kinetics have placed important constraints on ribosome structural dynamics. However, a gulf remains between static structures and time dependent data. In particular, X-ray crystallography and cryo-EM studies produce static models of the ribosome in various states, but lack dynamic information. Single molecule studies produce information on the rates of transitions between these states but do not have high-resolution spatial information. Computational studies have aided in bridging this gap by providing atomic resolution simulations of structural fluctuations and transitions between configurations. PMID:22336622

  4. Computational studies of molecular machines: the ribosome

    PubMed Central

    Sanbonmatsu, Karissa Y.

    2013-01-01

    The past decade has produced an avalanche of experimental data on the structure and dynamics of the ribosome. Groundbreaking studies in structural biology and kinetics have placed important constraints on ribosome structural dynamics. However, a gulf remains between static structures and time dependent data. In particular, x-ray crystallography and cryo-EM studies produce static models of the ribosome in various states, but lack dynamic information. Single molecule studies produce information on the rates of transitions between these states but do not have high-resolution spatial information. Computational studies have aided in bridging this gap by providing atomic resolution simulations of structural fluctuations and transitions between configurations. PMID:22336622

  5. A Competitive Nucleotide Binding Inhibitor: In vitro Characterization of Rab7 GTPase Inhibition

    PubMed Central

    Agola, Jacob O.; Hong, Lin; Surviladze, Zurab; Ursu, Oleg; Waller, Anna; Strouse, J. Jacob; Simpson, Denise S.; Schroeder, Chad E.; Oprea, Tudor I.; Golden, Jennifer E.; Aubé, Jeffrey; Buranda, Tione; Sklar, Larry A.; Wandinger-Ness, Angela

    2012-01-01

    Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (Ki) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase as well as serving as a model for other small molecular weight GTPase inhibitors. PMID:22486388

  6. Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes.

    PubMed

    Petibon, Cyrielle; Parenteau, Julie; Catala, Mathieu; Elela, Sherif Abou

    2016-05-01

    Most budding yeast introns exist in the many duplicated ribosomal protein genes (RPGs) and it has been posited that they remain there to modulate the expression of RPGs and cell growth in response to stress. However, the mechanism by which introns regulate the expression of RPGs and their impact on the synthesis of ribosomal proteins remain unclear. In this study, we show that introns determine the ratio of ribosomal protein isoforms through asymmetric paralog-specific regulation of splicing. Exchanging the introns and 3' untranslated regions of the duplicated RPS9 genes altered the splicing efficiency and changed the ratio of the ribosomal protein isoforms. Mutational analysis of the RPS9 genes indicated that splicing is regulated by variations in the intron structure and the 3' untranslated region. Together these data suggest that preferential splicing of duplicated RPGs provides a means for adjusting the ratio of different ribosomal protein isoforms, while maintaining the overall expression level of each ribosomal protein. PMID:26945043

  7. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis

    NASA Astrophysics Data System (ADS)

    Finley, Daniel; Bartel, Bonnie; Varshavsky, Alexander

    1989-03-01

    Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.

  8. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  9. Structure of ERA in Complex with the 3 End of 16s rRNBA Implications for Ribosome Biogenesis

    SciTech Connect

    Tu, C.; Zhou, X; Tropea, J; Austin, B; Waugh, D; Court, D; Ji, X

    2009-01-01

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3? end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  10. Illuminating Parasite Protein Production by Ribosome Profiling.

    PubMed

    Parsons, Marilyn; Myler, Peter J

    2016-06-01

    While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low-abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites including trypanosomes and Plasmodium. It has been used to identify new protein-coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation. PMID:27061497

  11. Marrow failure: a window into ribosome biology.

    PubMed

    Ruggero, Davide; Shimamura, Akiko

    2014-10-30

    Diamond-Blackfan anemia, Shwachman-Diamond syndrome, and dyskeratosis congenita are inherited syndromes characterized by marrow failure, congenital anomalies, and cancer predisposition. Genetic and molecular studies have uncovered distinct abnormalities in ribosome biogenesis underlying each of these 3 disorders. How defects in ribosomes, the essential organelles required for protein biosynthesis in all cells, cause tissue-specific abnormalities in human disease remains a question of fundamental scientific and medical importance. Here we review the overlapping and distinct clinical features of these 3 syndromes and discuss current knowledge regarding the ribosomal pathways disrupted in each of these disorders. We also explore the increasing complexity of ribosome biology and how this informs our understanding of developmental biology and human disease. PMID:25237201

  12. The immunological properties of Brucella ribosomal preparations.

    PubMed

    Corbel, M J

    1976-01-01

    Ribosomes were isolated from Brucella abortus strains 19 and 45/20 by disruption of the cells followed by differential ultracentrifugation. The ribosome preparations contained 2-3 components reacting in immunodiffusion tests but were free of detectable lipopolysaccharide-protein agglutinogen. They crossreacted with antisera to Br. abortus, Br. melitensis, Br. suis and Br. ovis and elicited intradermal delayed hypersensitivity reactions in animals infected with Br. abortus, Br. melitensis or Br. suis. The ribosomes were antigenic in rabbits, guinea pigs and mice. Those from Br. abortus S19 induced agglutinins reaction with smooth brucella strains whereas those from Br. abortus 45/20 induced agglutinins reacting with rough brucella strains. Cattle vaccinated with S19 or 45/20 vaccines or infected with Br. abortus developed pricipitins to ribosomal components at an early stage in the immune response. PMID:816681

  13. Marrow failure: a window into ribosome biology

    PubMed Central

    Ruggero, Davide

    2014-01-01

    Diamond-Blackfan anemia, Shwachman-Diamond syndrome, and dyskeratosis congenita are inherited syndromes characterized by marrow failure, congenital anomalies, and cancer predisposition. Genetic and molecular studies have uncovered distinct abnormalities in ribosome biogenesis underlying each of these 3 disorders. How defects in ribosomes, the essential organelles required for protein biosynthesis in all cells, cause tissue-specific abnormalities in human disease remains a question of fundamental scientific and medical importance. Here we review the overlapping and distinct clinical features of these 3 syndromes and discuss current knowledge regarding the ribosomal pathways disrupted in each of these disorders. We also explore the increasing complexity of ribosome biology and how this informs our understanding of developmental biology and human disease. PMID:25237201

  14. Ribosome Inactivating Proteins from Rosaceae.

    PubMed

    Shang, Chenjing; Rougé, Pierre; Van Damme, Els J M

    2016-01-01

    Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins. PMID:27556443

  15. Centres of excellence.

    PubMed

    Watson, J M

    1980-05-16

    The present Government may not be enthusiastic about health centres. But Dr Joyce M. Watson, of Glasgow University Department of General Practice and based at Woodside Health Centre in Glasgow, writes with enthusiasm of their advantages for the practice of medicine and the care of patients. PMID:10247174

  16. Frozen spin targets in ribosomal structure research.

    PubMed

    Stuhrmann, H B

    1991-01-01

    Polarized neutron scattering strongly depends on nuclear spin polarisation, particularly on proton spin polarisation. A single proton in a deuterated environment then is as efficient as 10 electrons in X-ray anomalous diffraction. Neutron scattering from the nuclear spin label is controlled by the polarisation of neutron spins and nuclear spins. Pure deuteron spin labels and proton spin labels are created by NMR saturation. We report on results obtained from the large subunit of E. coli ribosomes which have been obtained at the research reactor of GKSS using the polarized target facility developed by CERN. The nuclear spins were oriented with respect to an external field by dynamic nuclear polarisation. Proton spin polarisations of more than 80% were obtained in ribosomes at temperatures below 0.5 K. At T = 130 mK the relaxation time of the polarized target is one month (frozen spin target). Polarized small-angle neutron scattering of the in situ structure of rRNA and the total ribosomal protein (TP) has been determined from the frozen spin targets of the large ribosomal subunit, which has been deuterated in the TP and rRNA respectively. The results agree with those from neutron scattering in H2O/D2O mixtures obtained at room temperature. This is a necessary prerequisite for the planned determination of the in situ structure of individual ribosomal proteins and especially of that of ribosome bound mRNA and tRNAs. PMID:1720669

  17. Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

    PubMed

    Yan, Shuangquan; Xu, Mengmeng; Duan, Xiangke; Yu, Zhaoxiao; Li, Qiming; Xie, Longxiang; Fan, Xiangyu; Xie, Jianping

    2016-09-01

    The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators. PMID:27345061

  18. Multiple Roles of the Small GTPase Rab7.

    PubMed

    Guerra, Flora; Bucci, Cecilia

    2016-01-01

    Rab7 is a small GTPase that belongs to the Rab family and controls transport to late endocytic compartments such as late endosomes and lysosomes. The mechanism of action of Rab7 in the late endocytic pathway has been extensively studied. Rab7 is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors, thus also having implications on signal transduction. Several Rab7 interacting proteins have being identified leading to the discovery of a number of different important functions, beside its established role in endocytosis. Furthermore, Rab7 has specific functions in neurons. This review highlights and discusses the role and the importance of Rab7 on different cellular pathways and processes. PMID:27548222

  19. Chromosome segregation control by Escherichia coli ObgE GTPase.

    PubMed

    Foti, James J; Persky, Nicole S; Ferullo, Daniel J; Lovett, Susan T

    2007-07-01

    Escherichia coli cells depleted of the conserved GTPase, ObgE, show early chromosome-partitioning defects and accumulate replicated chromosomes in which the terminus regions are colocalized. Cells lacking ObgE continue to initiate replication, with a normal ratio of the origin to terminus. Localization of the SeqA DNA binding protein, normally seen as punctate foci, however, was disturbed. Depletion of ObgE also results in cell filamentation, with polyploid DNA content. Depletion of ObgE did not cause lethality, and cells recovered fully after expression of ObgE was restored. We propose a model in which ObgE is required to license chromosome segregation and subsequent cell cycle events. PMID:17578452

  20. Coevolution of RAC Small GTPases and their Regulators GEF Proteins

    PubMed Central

    Jiménez-Sánchez, Alejandro

    2016-01-01

    RAC proteins are small GTPases involved in important cellular processes in eukaryotes, and their deregulation may contribute to cancer. Activation of RAC proteins is regulated by DOCK and DBL protein families of guanine nucleotide exchange factors (GEFs). Although DOCK and DBL proteins act as GEFs on RAC proteins, DOCK and DBL family members are evolutionarily unrelated. To understand how DBL and DOCK families perform the same function on RAC proteins despite their unrelated primary structure, phylogenetic analyses of the RAC, DBL, and DOCK families were implemented, and interaction patterns that may suggest a coevolutionary process were searched. Interestingly, while RAC and DOCK proteins are very well conserved in humans and among eukaryotes, DBL proteins are highly divergent. Moreover, correlation analyses of the phylogenetic distances of RAC and GEF proteins and covariation analyses between residues in the interacting domains showed significant coevolution rates for both RAC–DOCK and RAC–DBL interactions. PMID:27226705

  1. Modulation of osteoclast differentiation and bone resorption by Rho GTPases

    PubMed Central

    Touaitahuata, Heiani; Blangy, Anne; Vives, Virginie

    2014-01-01

    Bone is a dynamic tissue constantly renewed through a regulated balance between bone formation and resorption. Excessive bone degradation by osteoclasts leads to pathological decreased bone density characteristic of osteolytic diseases such as post-menopausal osteoporosis or bone metastasis. Osteoclasts are multinucleated cells derived from hematopoietic stem cells via a complex differentiation process. Their unique ability to resorb bone is dependent on the formation of the actin-rich sealing zone. Within this adhesion structure, the plasma membrane differentiates into the ruffled border where protons and proteases are secreted to demineralize and degrade bone, respectively. On the bone surface, mature osteoclasts alternate between stationary resorptive and migratory phases. These are associated with profound actin cytoskeleton reorganization, until osteoclasts die of apoptosis. In this review, we highlight the role of Rho GTPases in all the steps of osteoclasts differentiation, function, and death and conclude on their interest as targets for treatment of osteolytic pathologies. PMID:24614674

  2. Functional Mapping of Human Dynamin-1-Like GTPase Domain Based on X-ray Structure Analyses

    PubMed Central

    Fröhlich, Chris; Eibl, Clarissa; Gimeno, Ana; Hessenberger, Manuel; Puehringer, Sandra; Daumke, Oliver; Goettig, Peter

    2013-01-01

    Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5′-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains. PMID:23977156

  3. New insights in the regulation of Rab GTPases by G protein-coupled receptors

    PubMed Central

    Lachance, Véronik; Angers, Stéphane; Parent, Jean-Luc

    2014-01-01

    Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, forming the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Growing evidence suggests that mutations, aberrant expression, and altered post-translational modifications of Rab GTPases are associated with human diseases. However, their regulatory mechanisms and how they are connected to cargo proteins are still poorly understood. Accumulating data indicate that G protein-coupled receptors (GPCRs) directly associate with Rab GTPases and that these interactions dictate receptor trafficking. Yet, it remained unclear whether the receptors could regulate the targeting and activity of Rab GTPases in various cell compartments. It is only in recent years that experimental studies showed that GPCR signaling and interaction with Rab-associated regulatory proteins modulate the localization and activity of Rab GTPases. This research is revealing novel regulatory mechanisms of these small GTPases and should contribute to the progress in effective drug development. Recently published in the Journal of Cell Science, Lachance et al. present a novel role for ubiquitylation of Rab11a by a β2AR/HACE1 complex in regulating Rab11a activity and β2AR trafficking. PMID:24950538

  4. Poliovirus protein 2C has ATPase and GTPase activities.

    PubMed

    Rodríguez, P L; Carrasco, L

    1993-04-15

    Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids. PMID:8385138

  5. Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

    PubMed Central

    García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

    2015-01-01

    Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

  6. ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

    PubMed Central

    East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

    2012-01-01

    The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

  7. The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

    SciTech Connect

    Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen; Bashan, Anat; Belousoff, Matthew; Wekselman, Itai; Zimmerman, Ella; Xiong, Liqun; Klepacki, Dorota; Arakawa, Kenji; Kinashi, Haruyasu; Mankin, Alexander S.; Yonath, Ada

    2010-04-26

    Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

  8. High-resolution structure of the Escherichia coli ribosome

    PubMed Central

    Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; Altman, Roger B.; Blanchard, Scott C.; Cate, Jamie H. D.

    2015-01-01

    Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. This structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development. PMID:25775265

  9. Mitochondrial ribosomal proteins (MRPs) of yeast.

    PubMed Central

    Graack, H R; Wittmann-Liebold, B

    1998-01-01

    Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host. Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter. Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae). To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher. Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences. Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described. The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research. Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans. PMID:9445368

  10. The ribosome triggers the stringent response by RelA via a highly distorted tRNA

    PubMed Central

    Agirrezabala, Xabier; Fernández, Israel S; Kelley, Ann C; Cartón, David Gil; Ramakrishnan, Venki; Valle, Mikel

    2013-01-01

    The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre. PMID:23877429