A direct estimation of the context effect on the efficiency of termination

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

doi:10.1006/jmbi.1998.2220

Cited by 57 publications

(49 citation statements)

References 27 publications

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“…RF1 was prepared from an overproducing strain as described by Dinçbas et al (16). RF2 overproduced from the wild-type gene is toxic to the cell and shows reduced activity in biochemical assays (17,18), especially with short peptidyl-tRNAs (our observations). RF2ala, which contains a threonine to alanine change at amino acid position 246, as in RF2 from Salmonella typhimurium (19), can be overproduced and yields a protein with enzymatic parameters close to those of RF2 prepared from normal cells: direct comparison with chromosomally produced RF2 shows that k cat is 2-fold lower.…”

Section: Methodssupporting

confidence: 57%

“…The effects of changing bases immediately downstream of stop codons on the termination efficiencies of RF1 and RF2 have been measured biochemically, and the largest context effect to be found is only a factor of 5 (18). This finding confirms that stop codons should be defined by their classical base triplets (23) and that nucleotide context can be responsible only for a fine-tuning of the termination reaction.…”

Section: Resultsmentioning

confidence: 65%

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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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152

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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: Methodssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 65%

The accuracy of codon recognition by polypeptide release factors

Freistroffer

Kwiatkowski

Buckingham

et al. 2000

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

Self Cite

152

150

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“…The RF-1 and RF-2 proteins are present in ϳ10-fold and 50-fold excess, respectively, over SsrA RNA in E. coli during log-phase growth (28). The affinities of these protein release factors for the A-site depends on the identities of the stop codon and following nucleotide (29). Because SsrA does not appear to have an anticodon stem-loop (30, 31), we assume that its affinity for the A-site is independent of the stop signal.…”

Section: Discussionmentioning

confidence: 99%

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

Hayes¹,

Bose

Sauer

2002

Journal of Biological Chemistry

145

177

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The SsrA or tmRNA quality control system relieves ribosome stalling and directs the addition of a degradation tag to the C terminus of the nascent chain. In some instances, SsrA tagging of otherwise full-length proteins occurs when the ribosome pauses at stop codons during normal translation termination. Here, the identities of the C-terminal residues of the nascent chain are shown to play an important role in full-length protein tagging. Specifically, a subset of C-terminal Xaa-Pro sequences caused SsrA tagging of the full-length YbeL protein from Escherichia coli. This tagging increased when a less efficient stop codon was used, increased in cells lacking protein release factor-3, and decreased when protein release factor-1 was overexpressed. Incorporation of the analog azetidine-2-carboxylic acid in place of proline suppressed tagging, whereas incorporation of 3,4-dehydroproline increased SsrA tagging of full-length YbeL. These results suggest that the detailed chemical or conformational properties of the C-terminal residues of the nascent polypeptide can affect the rate of translation termination, thereby influencing ribosome pausing and SsrA tagging at stop codons.

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“…As previously mentioned, tyrosine in itself has a negative gradient in H, complicating analysis of false termination errors. Given that +4 context increases the strength of termination (25,26), it is probable that release factor misreading may also be amplified by context. However, there are no particularly significant gradients in any of the +4 contexts.…”

Section: Near-stop Errorsmentioning

confidence: 99%

Gradients in nucleotide and codon usage along Escherichia coli genes

Hooper

Berg

2000

Nucleic Acids Research

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The usage of codons and nucleotide combinations varies along genes and systematic variation causes gradients in usage. We have studied such gradients of nucleotides and nucleotide combinations and their immediate context in Escherichia coli. To distinguish mutational and selectional effects, the genes were subdivided into three groups with different codon usage bias and the gradients of nucleotide usage were studied in each group. Some combinations that can be associated with a propensity for processivity errors show strong negative gradients that become weaker in genes with low codon bias, consistent with a selection on translational efficiency. One of the strongest gradients is for third position G, which shows a pervasive positive gradient in usage in most contexts of surrounding bases.

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A direct estimation of the context effect on the efficiency of termination

Cited by 57 publications

References 27 publications

The accuracy of codon recognition by polypeptide release factors

The accuracy of codon recognition by polypeptide release factors

Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

Gradients in nucleotide and codon usage along Escherichia coli genes

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