Escherichia coli tRNA(Phe) transcript lacking all the modified nucleosides was investigated in an in vitro translation system. To estimate the affinity of tRNA toward EF-Tu, Kd and K-1 were measured by the nuclease protection assay, and it was shown that the absence of modifications decreases ternary complex stability less than 2-fold. The activity of unmodified Phe-tRNA(Phe) on E. coli ribosomes was compared to modified Phe-tRNA(Phe) using the framework of the kinetic proofreading mechanism (Thompson & Dix, 1982) with both cognate and noncognate codons. Values of the individual rate constants in the elongation process showed that the modifications increased the accuracy of translation by (1) decreasing the rate of dipeptide synthesis and (2) increasing the rate of rejection with noncognate codons.
A sharp local existence and uniqueness theory for the initial-value problem for Burgers' equation is given in the Sobolev spaces H s , ?1=2 < s 0. It is proved that these results cannot be extended to any s < ?1=2 because uniqueness fails. A particular nontrivial solution is found which converges to 0 in the H s-norm as t ! 0 + .
Phe-tRNA (anticodon GAA)-polypeptidechain elongation factor Tu-GTP ternary complexes react faster with ribosomes programmed with UUC codons than with ribosomes programmed with UUU codons. A similar preference is shown by Leu-tRNA2 (anticodon GAG) complexes, which react faster with ribosomes programmed with CUC than with those programmed with CUU. The difference is seen in the rate of ternary-complex binding to the ribosome; no differences are seen in peptide-bond formation. Highly expressed mRNAs in Escherichia coli favor codons terminating in cytosine rather than uracil when both codons are read by a single tRNA with an anticodon beginning with guanine. The results suggest that intrinsic differences between the efficiencies of synonymous codons play an important role in modulating gene expression in E. coli.The genetic code is degenerate in that most amino acids may be specified by more than one codon. In the extreme case, incorporation of serine, arginine, or leucine may be directed by six different codons. Whether apparently synonymous codons behave differently in translation has been much debated; the choice of one codon over another might then modulate gene expression. That many organisms show a significant bias in the codons used in highly expressed genes (1, 2) supports this possibility. Indeed, there is direct evidence that codon choice can affect translation: replacing a rare leucine codon by a common one in the attenuator region of the leu operon of Salmonella typhimurium prevents attenuation (3) and replacing common codons with rare ones can reduce the amount of resulting protein (4). Pederson (5) and Varenne et al. (6) Evidence for some inherent specificity in the translational apparatus is provided by the finding of Parker and his colleagues (9) that Lys-tRNA is more likely to react with ribosomes programmed with the noncognate AAU codon (Asn) than with ribosomes programmed with the synonymous noncognate codon, AAC. However, demonstrating that cognate aa-tRNAs distinguish between ribosomes programmed with synonymous codons has been difficult. Recent advances in the synthesis of simple mRNAs and in techniques for measuring the kinetics of in vitro protein synthesis now allow us to measure in vitro the rate constants for the reaction of an aa-tRNA ternary complex (TC) with ribosomes programmed by synonymous codons. In this paper we show that synonymous codons do differ in the rate with which they direct ribosomes to react with the cognate aa-tRNA in vitro and that an intrinsic specificity for certain codons is built into the translational apparatus. In a parallel series of experiments J. Curran and M. Yarus (unpublished work) demonstrated that the same set of synonymous codons are translated at different rates in vivo. Therefore, the specificity we see in vitro is probably physiologically significant and could direct the evolution of codon bias.MATERIALS AND METHODS Sources of tRNAs, radioactive amino acids, and 32P-labeled inorganic phosphate have been described (7,10), except that tRNALeU...
Ribosomes programmed by different synonymous codons also differ in discriminating among near-cognate aminoacylated tRNAs. In the initial step of the recognition reaction ribosomes programmed by UUC discriminate less well than ribosomes programmed by UUU against ternary complexes containing three types of Leu-tRNA, and ribosomes programmed by CUC discriminate less well than ribosomes programmed by CUU against ternary complexes containing Phe-tRNA. Furthermore, in the proofreading step ribosomes programmed by UUC discriminate less well than ribosomes programmed by UUU against two of three near-cognate LeutRNAs, and ribosomes programmed by CUC discriminate less well than ribosomes programmed by CUU against nearcognate Phe-tRNA. The codon-induced change in reaction rate with near-cognate ternary complexes is greater than that with cognate ternary complexes: the most efficient codon is, therefore, the least accurate. Because the efficient, but inaccurate, codon UUC is used preferentially in highly expressed mRNAs of Escherichia coli, maxinmization of translational accuracy apparently has not been significant in the evolution of this particular biased codon choice in E. coli.The ribosomes, tRNAs, and factors of the translational apparatus are usually thought devoid of intrinsic specificity because any specificity could interfere with accurate reading of mRNA. But surprisingly the translational apparatus shows a distinct specificity for certain codon-tRNA pairs. The translational apparatus of many organisms appears to prefer a distinct subset of possible codons in highly expressed genes (1-4). Explanations for this bias based on the existence of preferred structures for mRNAs are unconvincing, and attempts to explain the bias now focuses on the hypothesis that the disfavored codons are either less rapidly or less accurately read by the translational apparatus. Evidence for the idea that disfavored codons are translated less rapidly includes the finding that the average in vivo translational-step times of highly expressed mRNAs increase when the mRNAs contain these codons (5) and that pause sites in translating several mRNAs associate with the disfavored codons (6). These findings have often been attributed to the fact that disfavored codons are generally translated by low-abundance tRNAs (7). However, we recently presented evidence that the ribosome works faster with certain codontRNA pairs (8). Thus, we showed in vitro that the phenylalanine codon UUC is more rapidly translated by Phe-tRNA than is the synonymous codon UUU. Results pointing to the same conclusion have been obtained in vivo by Curran and Yarus (29). That a favored codon is translated almost twice as fast as its disfavored synonym supports the idea that need for translational efficiency is one factor driving the evolution of codon bias in Escherichia coli.Another factor possibly contributing to the evolution of codon bias is the need for accurate translation. The choice of asparagine codons may provide an example of this effect. Parker and his co...
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