Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner

Freistroffer, David V.; Pavlov, Michael Y.; MacDougall, Jane; Buckingham, Richard H.; Ehrenberg, Måns

doi:10.1093/emboj/16.13.4126

Cited by 265 publications

(266 citation statements)

References 40 publications

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“…RF2 recognizes UAA and UGA with about equal efficiency (14), so it might have been expected that the efficiency with which the factor reads the sense codons AAA and AGA or GAA and GGA would be similar. In contrast, we find that RF2 becomes much more error-prone when G rather than A is present in the second position, although both dinucleotides AA and AG are part of the normal stop codons of the factor (Table 4).…”

Section: Resultsmentioning

confidence: 99%

“…RF1 normally terminates at UAA and UAG and RF2 at UAA and UGA (10). The dissociation of both factors from the ribosome after hydrolysis of the ester bond in peptidyl-tRNA connecting peptide chain with tRNA is accelerated by a third RF, the G-protein RF3 (11)(12)(13), and this seems so far to be its only function (14). The three stop codons together have about 20 sense codons as nearest neighbors (Tables 1 and 2), and hot spots for false stops are likely to be found within this set of sense triplets.…”

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confidence: 99%

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The accuracy of codon recognition by polypeptide release factors

Freistroffer

Kwiatkowski

Buckingham

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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The precision with which individual termination codons in mRNA are recognized by protein release factors (RFs) has been measured and compared with the decoding of sense codons by tRNA. An Escherichia coli system for protein synthesis in vitro with purified components was used to study the accuracy of termination by RF1 and RF2 in the presence or absence of RF3. The efficiency of factor-dependent termination at all sense codons differing from any of the three stop codons by a single mutation was measured and compared with the efficiency of termination at the three stop codons. RF1 and RF2 discriminate against sense codons related to stop codons by between 3 and more than 6 orders of magnitude. This high level of accuracy is obtained without energy-driven error correction (proofreading), in contrast to codon-dependent aminoacyl-tRNA recognition by ribosomes. Two codons, UAU and UGG, stand out as hotspots for RF-dependent premature termination.protein synthesis ͉ translational processivity ͉ premature termination S ixty-one of the 64 base triplets in the genetic code are sense codons, which are translated in bacteria to the 20 standard amino acids in proteins by about 50 aminoacyl-tRNAs. The remaining three codons usually signal termination of translation and the release of completed proteins from ribosomes by release factors RF1 and RF2. Efficient translation requires both rapid binding to their respective codons of cognate aminoacyl-tRNAs, in ternary complex with elongation factor EF-Tu and GTP, and fast termination of protein synthesis at stop signals by RFs. In addition to fast signal processing, codon translation must attain a high level of accuracy so that nonacceptable amino acid replacements in nascent proteins do not impair cell growth and viability. Similarly, misrecognition by RFs of sense codons as stop signals leads to truncated proteins, an energetically costly event, the frequency of which also must be contained.Pauling was among the first to reflect on the errors that must occur during protein synthesis because of the limited maximum differences in interaction standard free energies between different pairs of amino acids, and he concluded that amino acid substitutions theoretically should occur at frequencies in the percent range (1). Though later shown to be overly pessimistic (2), these predictions led to theoretical studies showing that enzymatic selection can, under certain conditions, be more accurate than the instrinsic selectivity of a single step.The accuracy (A) of simple enzyme selection schemes, defined as the probability of correct product formation divided by the probability of an error at equal concentrations of cognate and noncognate substrates, is limited by the standard free energy difference between the transition state leading to product formation and ground state for cognate (⌬G c ) and noncognate (⌬G nc ) substrates by the inequality. The precision of codon translation depends on differences in H-bond energies between cognate and noncognate tRNAcodon interactions, though other i...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

The accuracy of codon recognition by polypeptide release factors

Freistroffer

Kwiatkowski

Buckingham

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

149

150

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“…Macrolides that do not reach the PT center allow formation of longer peptides, and erythromycin, which belongs to this sub-class, was chosen as the other model compound. The mechanisms of action of josamycin and erythromycin were studied in a cellfree translation system, reconstituted from pure E. coli components (7,17).…”

Section: Discussionmentioning

confidence: 99%

“…In this study we have clarified some of these issues for the macrolides erythromycin and josamycin by studying their kinetic properties in a cell-free system for protein synthesis with Escherichia coli components of high purity (7,17).…”

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confidence: 99%

Kinetics of Macrolide Action

Lovmar

Tenson

Ehrenberg

2004

Journal of Biological Chemistry

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Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nM, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of fulllength proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacteriotoxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyltRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.

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“…Ehrenberg and his co-workers have reported that Escherichia coli RF3 accelerates the dissociation of the release factors RF1 and RF2 from the ribosome in a GTP-dependent manner and that fast recycling of ribosomes requires both RF3 and RRF [6,7]. Grentzmann et al [8] have observed similar results and found that RF3 can substitute for EF-G in RRFdependent ribosome recycling reactions in vitro.…”

Section: Introductionmentioning

confidence: 87%

Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C‐terminal modulator element

et al. 1999

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Ribosome recycling factor, referred to as RRF, is essential for bacterial growth because of its activity of decomposition of the post-termination complex of the ribosome after release of polypeptides. In this study, we isolated a conditionally lethal amber mutation, named frr-3, in the Escherichia coli RRF gene at amino acid position 161, showing that the truncation of the C-terminal 25 amino acids of RRF is lethal to E. coli. An RRF gene cloned from Thermus thermophilus, whose protein is 44% identical and 68% similar to E. coli RRF, failed to complement the frr-3(Am) allele. However, truncation of the C-terminal five amino acids conferred intergeneric complementation activity on T. thermophilus RRF, demonstrating the modulator activity of the C-terminal tail. Rapid purification of T. thermophilus RRF was achieved by T7-RNA polymerase-driven overexpression for crystallography.z 1999 Federation of European Biochemical Societies.

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Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner

Cited by 265 publications

References 40 publications

The accuracy of codon recognition by polypeptide release factors

The accuracy of codon recognition by polypeptide release factors

Kinetics of Macrolide Action

Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C‐terminal modulator element

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