Elongation Factor Tu Research Articles

Conserved Discrimination against Misacylated tRNAs by Two Mesophilic Elongation Factor Tu Orthologs

Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.

The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci

Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions.

Metabolic Characteristic of the Liver of Dairy Cows during Ketosis Based on Comparative Proteomics

The objective of the present study was to identify differences in the expression levels of liver proteins between healthy and ketotic cows, establish a liver metabolic interrelationship of ketosis and elucidate the metabolic characteristics of the liver during ketosis. Liver samples from 8 healthy multiparous Hostein cows and 8 ketotic cows were pooled by health status and the proteins were separated by two-dimensional-electrophoresis (2D-E). Statistical analysis of gels was performed using PDQuest software 8.0. The differences in the expression levels of liver proteins (p<0.05) between ketotic and healthy cows were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry. Five enzymes/proteins were identified as being differentially expressed in the livers of ketotic cows: expression of 3-hydroxyacyl-CoA dehydrogenase type-2 (HCDH), acetylcoenzyme A acetyltransferase 2 (ACAT) and elongation factor Tu (EF-Tu) were down-regulated, whereas that of alpha-enolase and creatine kinase were up-regulated. On the basis of this evidence, it could be presumed that the decreased expression of HCDH, which is caused by high concentrations of acetyl-CoA in hepatic cells, in the livers of ketotic cows, implies reduced fatty acid β-oxidation. The resultant high concentrations of acetyl-CoA and acetoacetyl CoA would depress the level of ACAT and generate more β-hydroxybutyric acid; high concentrations of acetyl-CoA would also accelerate the Krebs Cycle and produce more ATP, which is stored as phosphocreatine, as a consequence of increased expression of creatine kinase. The low expression level of elongation factor Tu in the livers of ketotic cows indicates decreased levels of protein synthesis due to the limited availability of amino acids, because the most glucogenic amino acids sustain the glyconeogenesis pathway; thus increasing the level of alpha-enolase. Decreased protein synthesis also promotes the conversion of amino acids to oxaloacetate, which drives the Krebs Cycle under conditions of high levels of acetyl-CoA. It is concluded that the livers of ketotic cows possess high concentrations of acetyl-CoA, which through negative feedback inhibited fatty acid oxidation; show decreased fatty acid oxidation, ketogenesis and protein synthesis; and increased gluconeogenesis and energy production.

Surface-bound proteins of Lactobacillus plantarum 423 that contribute to adhesion of Caco-2 cells and their role in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis

Elongation factor Tu (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are surface-bound proteins with a role in adhesion of Lactobacillus plantarum 423 to Caco-2 cells. Removal of surface-bound proteins from L. plantarum 423 (treated with 4 M guanidine–HCl) reduced adhesion to Caco-2 cells by 40%. In a competitive exclusion experiment where all three strains were given an equal chance to adhere to Caco-2 cells, L. plantarum 423 prevented 71% of cells of Clostridium sporogenes LMG 13570 and 89% of cells of Enterococcus faecalis LMG 13566 from adhering. Cells of L. plantarum 423, from which surface-bound proteins were removed, prevented 49% of cells of C. sporogenes LMG 13570 and 70% of cells of E. faecalis LMG 13566 from adhering to Caco-2 cells, suggesting that factors other than surface-bound proteins are involved in adhesion. Colonization of L. plantarum 423 to Caco-2 cells prevented adhesion of 74% of cells of C. sporogenes LMG 13570 and 62% of cells of E. faecalis LMG 13566. Furthermore, L. plantarum 423 displaced 81% of cells of C. sporogenes LMG 13570 and 91% of cells of E. faecalis LMG 13566 from Caco-2 cells. L. plantarum 423 is a potential probiotic strain.

Polyelectrolyte Behavior and Kinetics of Aminoacyl-tRNA on the Ribosome

The torque acting on cognate (three base pairs that are matched) "ternary complex" consisting of elongation factor-Tu, guanosine-5'-triphosphate GTP, and aminoacyl-transfer RNA due to induced wrapping of the 30S subunit of the ribosome and the speed with which the ternary complex samples the space allowed by diffusion is determined. Under appropriate conditions, mode coupling speeds up the barrier crossing rate for cognate relative to near-cognate ternary complexes. We determine the flexibility of the ternary complex relative to transfer RNA (tRNA) by a coarse-grained model. We predict the magnesium binding sites in the ternary complex at low magnesium concentration and unravel the nature of the interaction energy of magnesium with site-specific tRNAPhe bases.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

Dynamics of Recognition between tRNA and Elongation Factor Tu

Elongation factor Tu (EF-Tu) binds to all standard aminoacyl transfer RNAs (aa-tRNAs) and transports them to the ribosome while protecting the ester linkage between the tRNA and its cognate amino acid. We use molecular dynamics simulations to investigate the dynamics of the EF-Tu·guanosine 5′-triphosphate·aa-tRNA Cys complex and the roles played by Mg 2+ ions and modified nucleosides on the free energy of protein·RNA binding. Individual modified nucleosides have pronounced effects on the structural dynamics of tRNA and the EF-Tu·Cys-tRNA Cys interface. Combined energetic and evolutionary analyses identify the coevolution of residues in EF-Tu and aa-tRNAs at the binding interface. Highly conserved EF-Tu residues are responsible for both attracting aa-tRNAs as well as providing nearby nonbonded repulsive energies that help fine-tune molecular attraction at the binding interface. In addition to the 3′ CCA end, highly conserved tRNA nucleotides G1, G52, G53, and U54 contribute significantly to EF-Tu binding energies. Modification of U54 to thymine affects the structure of the tRNA common loop resulting in a change in binding interface contacts. In addition, other nucleotides, conserved within certain tRNA specificities, may be responsible for tuning aa-tRNA binding to EF-Tu. The trend in EF-Tu·Cys-tRNA Cys binding energies observed as the result of mutating the tRNA agrees with experimental observation. We also predict variations in binding free energies upon misacylation of tRNA Cys with d-cysteine or O-phosphoserine and upon changing the protonation state of l-cysteine. Principal components analysis in each case reveals changes in the communication network across the protein·tRNA interface and is the basis for the entropy calculations.

High-throughput identification of T-lymphocyte antigens from Anaplasma marginale expressed using in vitro transcription and translation

The ability to rapidly screen a complex pathogen proteome for proteins that elicit recall T-lymphocyte responses from immune individuals would accelerate vaccine development. An outer membrane fraction of the rickettsial pathogen Anaplasma marginale induces protective immunity against infection and disease in cattle. We have used this immunization model to evaluate high-throughput screening of proteins expressed by in vitro transcription and translation (IVTT) for recognition by memory CD4 + T-lymphocytes. Fifty selected vaccine candidate antigens identified from the A. marginale genome were expressed from transcriptionally active PCR products using an Escherichia coli-based IVTT system, and bead-affinity purified using antibodies to His and FLAG epitope tags. IVTT-expressed bead-bound antigens were processed and presented by antigen presenting cells to T-lymphocytes from outer membrane immunized animals and evaluated for immunogenicity in proliferation assays. Antigens that consistently stimulated responses were known T-cell antigens major surface protein (MSP)2, MSP3, VirB9, and VirB10 and newly identified T-cell antigens outer membrane protein (OMP)4, OMP9, elongation factor-Tu, Ana29, and OMA87. Specific T-cell stimulation was achieved even at low antigen concentration, and was highly sensitive when compared with unbound IVTT reaction products. This method allows rapid expression and identification of T-lymphocyte antigens for any pathogen for which the genome sequence is available.

Modification of the Ribosome and the Translational Machinery during Reduced Growth Due to Environmental Stress.

Escherichia coli strains normally used under laboratory conditions have been selected for maximum growth rates and require maximum translation efficiency. Recent studies have shed light on the structural and functional changes undergone by the translational machinery in E. coli during heat and cold shock and upon entry into stationary phase. In these situations both the composition and the partitioning of this machinery into the different pools of cellular ribosomes are modified. As a result, the translational capacity of the cell is dramatically altered. This review provides a comprehensive account of these modifications, regardless of whether or not their underlying mechanisms and their effects on cellular physiology are known. Not only is the composition of the ribosome modified upon entry into stationary phase, but the modification of other components of the translational machinery, such as elongation factor Tu (EFTu) and tRNAs, has also been observed. Hibernation-promoting factor (HPF), paralog protein Y (PY), and ribosome modulation factor (RMF) may also be related to the general protection against environmental stress observed in stationary-phase E. coli cells, a role that would not be revealed necessarily by the viability assays. Even for the best-characterized ribosome-associated factors induced under stress (RMF, PY, and initiation factors), we are far from a complete understanding of their modes of action.

Salinity-induced physiological and proteomic changes in Anabaena doliolum

This study is the first to offer information on salinity-induced inhibition of physiological variables, changes in proteome, and induction of glycolate metabolism in Anabaena doliolum. A significant reduction in O 2-evolution, carbon fixation, chlorophyll and NADPH/NADH level and increase in intracellular Na + and respiration were observed following 150 mM NaCl treatment for 1 and 24 h. Interestingly, ATP content registered significant decrease after 1 h and recovery after 24 h treatment of 150 mM NaCl. Two-dimensional gel electrophoresis and MALDI-TOF MS detected a set of six proteins showing significant reproducible alterations, and homology with iron superoxide dismutase, superoxide dismutase (imported), phycocyanin alpha chain, elongation factor-Tu (EF-Tu), ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase of Nostoc PCC7120. Increased RuBisCO and decreased carbon fixation suggested operation of glycolate metabolism. This was confirmed by accumulation of free and phospho-glyceric acid, increase in glycolate oxidase activity, glycine, serine and ammonium contents. Since peroxide generated in this pathway cannot be scavenged due to sensitivity of catalase to NaCl the organism fails to acclimatize under salt stress.

A novel receptor – ligand pathway for entry of Francisella tularensis in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu

BackgroundFrancisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested.ResultsWe show here that cell-surface expressed nucleolin is a receptor for Francisella tularensis Live Vaccine Strain (LVS) and promotes LVS binding and infection of human monocyte-like THP-1 cells. The HB-19 pseudopeptide that binds specifically carboxy-terminal RGG domain of nucleolin inhibits LVS binding and infection of monocyte-like THP-1 cells. In a pull-down assay, elongation factor Tu (EF-Tu), a GTP-binding protein involved in protein translation, usually found in cytoplasm, was recovered among LVS bacterial membrane proteins bound on RGG domain of nucleolin. A specific polyclonal murine antibody was raised against recombinant LVS EF-Tu. By fluorescence and electron microscopy experiments, we found that a fraction of EF-Tu could be detected at the bacterial surface. Anti-EF-Tu antibodies reduced LVS binding to monocyte-like THP-1 cells and impaired infection, even in absence of complement and complement receptors. Interaction between EF-Tu and nucleolin was illustrated by two different pull-down assays using recombinant EF-Tu proteins and either RGG domain of nucleolin or cell solubilized nucleolin.DiscussionAltogether, our results demonstrate that the interaction between surface nucleolin and its bacterial ligand EF-Tu plays an important role in Francisella tularensis adhesion and entry process and may therefore facilitate invasion of host tissues. Since phagosomal escape and intra-cytosolic multiplication of LVS in infected monocytes are very similar to those of human pathogenic F. tularensis ssp tularensis, the mechanism of entry into monocyte-like THP-1 cells, involving interaction between EF-Tu and nucleolin, might be similar in the two subspecies. Thus, the use of either nucleolin-specific pseudopeptide HB-19 or recombinant EF-Tu could provide attractive therapeutic approaches for modulating F. tularensis infection.

Elongation Factor Tu Mutants Expand Amino Acid Tolerance of Protein Biosynthesis System

Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E. coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2-anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.

Thiostrepton inhibition of tRNA delivery to the ribosome

Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.

Phenylalanyl-tRNA synthetase editing defects result in efficient mistranslation of phenylalanine codons as tyrosine

Translational quality control is monitored at several steps, including substrate selection by aminoacyl-tRNA synthetases (aaRSs), and discrimination of aminoacyl-tRNAs by elongation factor Tu (EF-Tu) and the ribosome. Phenylalanyl-tRNA synthetase (PheRS) misactivates Tyr but is able to correct the mistake using a proofreading activity named editing. Previously we found that overproduction of editing-defective PheRS resulted in Tyr incorporation at Phe-encoded positions in vivo, although the misreading efficiency could not be estimated. This raised the question as to whether or not EF-Tu and the ribosome provide further proofreading mechanisms to prevent mistranslation of Phe codons by Tyr. Here we show that, after evading editing by PheRS, Tyr-tRNA(Phe) is recognized by EF-Tu as efficiently as the cognate Phe-tRNA(Phe). Kinetic decoding studies using full-length Tyr-tRNA(Phe) and Phe-tRNA(Phe), as well as a poly(U)-directed polyTyr/polyPhe synthesis assay, indicate that the ribosome lacks discrimination between Tyr-tRNA(Phe) and Phe-tRNA(Phe). Taken together, these data suggest that PheRS editing is the major proofreading step that prevents infiltration of Tyr into Phe codons during translation.

BAK1 for Pathogen Response

BAK1 (brassinosteroid insensitive 1-associated receptor kinase 1) is best known and is named for its role as a leucine-rich repeat receptor-like kinase (LRR-RLK) involved in brassinosteroid signaling in Arabidopsis thaliana . Chinchilla et al . report that BAK1 also contributes to the response to pathogen-associated molecular patterns (PAMPs), such as those triggered by bacterial flagellin or elongation factor Tu (EF-Tu). Heese et al . identified BAK1 as a coimmunoprecipitation partner for Arabipdosis FLS2. Chinchilla et al . identified plants with insertions in the bak1 gene in a screen for genes that encode LRR-RLKs that produce plants with decreased sensitivity to the PAMP flagellin peptide flg22 (the growth of seedlings was not inhibited by flg22). The insertional mutant plants also showed decreased responsiveness to brassinosteroids; however, the defect in brassinosteroid signaling was not the cause of the defective PAMP response. Even when brassinosteroid signaling was restored by high concentrations of exogenous brassinolide, the bak1 mutant seedlings failed to exhibit growth inhibition in response to flg22. Both groups reported that stimulation of mitogen-activated protein kinase activity and the oxidative burst that are characteristic of the wild-type response to flg22 were diminished in bak1 mutant plants. Both groups also determined that BAK1 was not specific to the flagellin PAMP but appeared to play a role in triggering responses to various PAMPs. Heese et al . identified the Nicotiana benthamiana homolog of BAK1 and found that knockdown of the protein in this plant allowed enhanced growth of bacteria on the plants. Thus, BAK1 appears to be a promiscuous LRR-RLK, partnering with various ligand-binding receptors to trigger ligand-specific responses. D. Chinchilla, C. Zipfel, S. Robatzek, B. Kemmerling, T. Nürnberger, J. D. G. Jones, G. Felix, T. Boller, A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448 , 497-500 (2007). [PubMed] A. Heese, D. R. Hann, S. Gimenez-Ibanez, A. M. E. Jones, K. He, J. Li, J. I. Schroeder, S. C. Peck, J. P. Rathjen, The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc. Natl. Acad. Sci. U.S.A. 104 , 12217-12222 (2007). [Abstract] [Full Text]

A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence

Plants sense potential microbial invaders by using pattern-recognition receptors to recognize pathogen-associated molecular patterns (PAMPs). In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref. 2) and elongation factor Tu receptor (EFR) (ref. 3) act as pattern-recognition receptors for the bacterial PAMPs flagellin and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucine-rich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellin-triggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.

The 51–63 base pair of tRNA confers specificity for binding by EF-Tu

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.

Kinetic Analysis of the Entire RNA Amplification Process by Qβ Replicase

The kinetics of the RNA replication reaction by Qbeta replicase were investigated. Qbeta replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qbeta and plays a key role in the life cycle of the Qbeta phage. Although the RNA replication reaction using this enzyme has long been studied, a kinetic model that can describe the entire RNA amplification process has yet to be determined. In this study, we propose a kinetic model that is able to account for the entire RNA amplification process. The key to our proposed kinetic model is the consideration of nonproductive binding (i.e. binding of an enzyme to the RNA where the enzyme cannot initiate the reaction). By considering nonproductive binding and the notable enzyme inactivation we observed, the previous observations that remained unresolved could also be explained. Moreover, based on the kinetic model and the experimental results, we determined rate and equilibrium constants using template RNAs of various lengths. The proposed model and the obtained constants provide important information both for understanding the basis of Qbeta phage amplification and the applications using Qbeta replicase.

Mechanism of EF-Ts-Catalyzed Guanine Nucleotide Exchange in EF-Tu: Contribution of Interactions Mediated by Helix B of EF-Tu

Elongation factor Tu (EF-Tu) belongs to the family of GTP-binding proteins and requires elongation factor Ts (EF-Ts) for nucleotide exchange. Crystal structures suggested that one of the salient features in the EF-Tu x EF-Ts complex is a conformation change in the switch II region of EF-Tu that is initiated by intrusion of Phe81 of EF-Ts between His84 and His118 of EF-Tu and may result in a destabilization of Mg2+ coordination and guanine nucleotide release. In the present paper, the contribution of His84 to nucleotide release was studied by pre-steady-state kinetic analysis of nucleotide exchange in mutant EF-Tu in which His84 was replaced by Ala. Both intrinsic and EF-Ts-catalyzed nucleotide release was affected by the mutation, resulting in a 10-fold faster spontaneous GDP release and a 4-fold faster EF-Ts-catalyzed release of GTP and GDP. Removal of Mg2+ from the EF-Tu x EF-Ts complex increased the rate constant of GDP release 2-fold, suggesting a small contribution to nucleotide exchange. Together with published data on the effects of mutations interfering with other putative interactions between EF-Tu and EF-Ts, the results suggest that each of the contacts in the EF-Tu x EF-Ts complex alone contributes moderately to nucleotide destabilization, but together they act synergistically to bring about the overall 60,000-fold acceleration of nucleotide exchange in EF-Tu by EF-Ts.

Structure probing of tmRNA in distinct stages of trans-translation

Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helper protein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work we used lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery at different steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reacted with Ala-tmRNA*EF-Tu*GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was probed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we also analyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. We observed some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNA conformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA is in complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.