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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two genes, tufA and tufB, located at 73 and 88 minutes of the Escherichia coli linkage map, code for the polypeptide chain elongation factor EF-Tu. tufB is transcribed with four upstream tRNA genes, thrU, tyrU, glyT and thrT, into a cotranscript of approximately 1800 nucleotides. Here we show that in vivo processing yields a 1320 nucleotide transcript of tufB. S1 nuclease fine mapping reveals that the processing site is located in the intergenic region at about 72 to 74 nucleotides upstream from the initiation codon of the tufB cistron. A deletion in the cloned tRNA-tufB operon, encompassing the 3' half of thrU, the complete tyrU, glyT, thrT genes and ten nucleotides of the intergenic region, causes a threefold increase of the rate of plasmid tufB transcription, a fourfold increase of plasmid-borne tufB RNA and a twofold increase of plasmid-borne EF-TuB. We conclude that the deletion has eliminated a transcription termination site probably located after the thrT gene. Termination at this site uncouples tRNA synthesis from tufB transcription.
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PMID:The tRNA-tufB operon transcription termination and processing upstream from tufB. 244 75

Translational frameshifts, both +1 and -1, are promoted by mutations in tufA and tufB, the two genes encoding the polypeptide chain elongation factor (EF) Tu of Escherichia coli. Strains harboring the mutant EF-Tu(Ala375----Thr) encoded by either tufA or tufB or by both, display a linear relationship between the frequency of frameshifting and the concentration of mutant EF-Tu, relative to the total amount of EF-Tu. A second mutant species, EF-TuB(Gly222----Asp), also promotes frameshifting. The frequency is strikingly enhanced by the combined action of EF-TuA(Ala375----Thr) and EF-TuB(Gly222----Asp) and exceeds by far the total contribution of the two mutant EF-Tus studied separately. These observations raise the question whether the formation of each peptide bond under conditions that no frameshifting occurs also requires the combined action of two EF-Tu molecules, in this case not differing functionally.
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PMID:Translational frameshifts induced by mutant species of the polypeptide chain elongation factor Tu of Escherichia coli. 266 15

The elongation factor EF-Tu of E. coli is a multifunctional protein that lends itself extremely well to studies concerning structure-function relationships. It is encoded by two genes: tufA and tufB. Mutant species of EF-Tu have been obtained by various genetic manipulations, including site- and segment-directed mutagenesis of tuf genes on a vector. The presence of multiple tuf genes in the cell, both chromosomal and plasmid-borne, hampers the characterization of the mutant EF-Tu. We describe a procedure for transferring plasmid-borne tuf gene mutations to the chromosome. Any mutation engineered by genetic manipulation of tuf genes on a vector can be transferred both to the tufA and the tufB position on the chromosome. The procedure facilitated the functional characterization of some of our recently obtained tuf mutations. Of particular relevance is, that it enabled us for the first time to obtain a mutant tufB on the chromosome, encoding an EF-TuB resistant to kirromycin. It thus became possible to study the consequences for growth of tufA inactivation by insertion of bacteriophage Mu. The preliminary evidence obtained suggests that an EF-TuA, active in polypeptide synthesis, is essential for growth whereas such an EF-TuB is dispensable.
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PMID:Transfer of plasmid-borne tuf mutations to the chromosome as a genetic tool for studying the functioning of EF-TuA and EF-TuB in the E. coli cell. 296 74

The polypeptide chain elongation factor EF-Tu of Escherichia coli is encoded by two genes, tufA and tufB, located in two different operons. Experiments in which either tufA or tufB was inactivated demonstrated that expression of the tRNA-tufB operon is dependent on a functioning tufA and thus on EF-Tu (1, to be published). In order to study a possible role of EF-Tu as trans-activator of the tRNA-tufB operon, we have investigated in vitro binding of an EF-Tu. GDP preparation to various DNA fragments of the operon. We demonstrate that specific binding occurs to a cis-acting region delimited from position -134 to the promoter, previously shown to enhance tufB transcription. Electrophoretic retardation assays reveal the formation of maximally three protein/DNA complexes, indicating that more than one protein molecule can bind to the DNA. The EF-Tu preparation used was obtained by affinity chromatography and appeared to be 95% pure. It lost its DNA binding activity upon further purification. That EF-Tu is nonetheless involved in the DNA binding is suggested by the observation that none of the three complexes is formed in the presence of kirromycin, an antibiotic that binds EF-Tu with high specificity. If so, EF-Tu.GDP most likely binds to the activator region of the tRNA-tufB operon in combination with another non-identified protein or component.
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PMID:The elongation factor EF-Tu from E. coli binds to the upstream activator region of the tRNA-tufB operon. 305 39

A new method (T. Ruusala et al., 1982, EMBO J. 1, 75-78, 741-748) for analyzing kinetic proofreading in translation is described. An in vitro system is arranged so that its rate of polypeptide synthesis is determined by the release rate of GDP from EF-Tu in the absence of EF-Ts. This enables the counting of the number of EF-Tu cycles for correct as well as for incorrect peptide bonds. The necessary equations are derived and the approximations involved in these are discussed together with data from experiments not previously described.
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PMID:Counting cycles of EF-Tu to measure proofreading in translation. 308 18

The binding of Phe-tRNAPhe at the programmed ribosomal A site has been investigated using antibiotics that influence this binding in different ways. The adhesion of Phe-tRNAPhe, the consumption of GTP and the extent of the peptidyl transfer reaction were monitored. All of the five known misreading-inducing antibiotics that were tested stabilised the binding of Phe-tRNAPhe after its affixture to the A site by EF-Tu with GTP hydrolysis. The stabilisation was sufficient to overcome a single mismatch in the codon-anticodon interaction. Combinations of stabilising and destabilising influences were found to be additive, thus supporting the concepts: (1) that there is a 'correct' binding energy for aminoacyl tRNA in the A site, whose reduction hampers polypeptide synthesis and whose increase makes it inaccurate by by-passing proofreading; and (2) that the different antibiotics affect the bound aminoacyl tRNA at different points.
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PMID:Decoding at the ribosomal A site: antibiotics, misreading and energy of aminoacyl-tRNA binding. 312 44

Erythromycin (a 14-membered macrolide) and virginiamycin S (a type B synergimycin) block protein biosynthesis in bacteria, but are virtually inactive on poly(U)-directed poly(Phe) synthesis. We have recently shown, however, that these antibiotics inhibit the in vitro polypeptide synthesis directed by synthetic copolymers: this effect is analyzed further in the present work. We were unable to find any consistent alteration produced by these antibiotics on coupled and uncoupled EF-G- and EF-Tu-dependent GTPases, on the EF-Tu-directed binding of aminoacyl-tRNA to ribosomes, and on the EF-G- and GTP-mediated translocation of peptidyl-tRNA bound to poly(U,C).ribosome complexes. With these complexes, the peptidyl transfer reaction, as measured by peptidylpuromycin synthesis, was 10-30% inhibited by virginiamycin S and erythromycin. A direct relationship between the virginiamycin S- and erythromycin-promoted inhibition of poly(A,C)-directed polypeptide synthesis, on the one hand, and the EF-G concentration and the rate of the polymerization reaction, on the other hand, was observed, in agreement with a postulated reversible inhibitor action of these antibiotics. The increased inhibitory activity, which was observed during the first 4-6 rounds of elongation, in the presence of virginiamycin S or erythromycin, was suggestive of a specific action of these antibiotics on the correct positioning of peptidyl-tRNA at the P site. The marked stimulation of premature release of peptidyl-tRNA from poly(A,C).ribosome complexes can be referred to an altered interaction of the C-terminal aminoacyl residue of the growing peptidyl chain with the ribosome. We conclude that the action of virginiamycin S and erythromycin entails a template-dependent alteration of the interaction of peptidyl-tRNA with the donor site of peptidyltransferase, which may lead to a transient functional block of the ribosome and in some instances to a premature release of peptidyl-tRNA and termination of the elongation process.
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PMID:Action of erythromycin and virginiamycin S on polypeptide synthesis in cell-free systems. 314 22

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.
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PMID:Extracellular release of colicin A is non-specific. 331 27

We have studied the consequences of mutational alteration in the structure of EF-Tu on the missense errors and proofreading activity of bacterial ribosomes in vitro. Our data show that the EF-Tu Bo mutant form of EF-Tu (van der Meide et al. 1983a) is inactive in polypeptide synthesis on the ribosome, even though it binds aminoacyl-tRNA. A second mutant form, EF-Tu Ar (van der Meide et al. 1983a), is active in polypeptide synthesis but supports a much higher messense incorporation with either leucine isoacceptor 2 or leucine isoacceptor 4 in the in vitro system. Further analysis of the kinetic basis of this enhanced missense frequency revealed that the mutation responsible for the alteration in EF-Tu Ar increases the errors at both the proofreading step and the initial selection. In this respect the effect of this particular mutation is similar to the mode of action of the antibiotic kanamycin (Jelenc and Kurland 1984).
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PMID:Mutant EF-Tu increases missense error in vitro. 354 May 29

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from the yeast Saccharomyces cerevisiae using a large-scale procedure. The three steps of purification used were batch adsorption on phosphocellulose, phosphocellulose chromatography and, as the last step, GDP-Sepharose or Biorex column chromatography. The protein is very basic (pI = 9.2) and has an apparent molecular mass of 49 kDa, as determined by polyacrylamide gel electrophoresis using denaturing conditions. It is one of the most abundant proteins in yeast (about 5% of total soluble protein), as shown by two-dimensional gel electrophoresis and by immunological titration. A strong immunological and structural homology was found between yeast EF-1 alpha and elongation factors from other sources. Common immunological features were found between yeast and wheat germ EF-1 alpha. Tryptic hydrolysis of yeast EF-1 alpha in the presence of 25% glycerol generated a large trypsin-resistant polypeptide (Mr = 43,000) which had the same NH2-terminal sequence as the proteolyzed product from rabbit reticulocyte, Artemia salina EF-1 alpha and Escherichia coli EF-Tu. Completed DNA sequence determination of one structural gene for yeast EF-1 alpha confirmed a remarkable conservation of several protein sequence domains in yeast and animal EF-1 alpha (Cottrelle, P., Thiele, D., Price, V., Memet, S., Micouin, J.Y., Marck, C., Buhler, J.M. Sentenac, A., and Fromageot, P. (1985) J. Biol. Chem. 260, 3090-3096).
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PMID:Elongation factor 1 alpha from Saccharomyces cerevisiae. Rapid large-scale purification and molecular characterization. 388 5


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