Biomedical subjects

D B Dix

Publications and source records attributed to D B Dix.

5 recordsLinked to original sources

PubMed1989

Codon choice and gene expression: synonymous codons differ in translational accuracy.

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 Leu-tRNAs, and ribosomes programmed by CUC discriminate less well than ribosomes programmed by CUU against near-cognate 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, maximization of translational accuracy apparently has not been significant in the evolution of this particular biased codon choice in E. coli.

Anticodon↗

PubMed1988

Codon choice and gene expression: synonymous codons differ in their ability to direct aminoacylated-transfer RNA binding to ribosomes in vitro.

Phe-tRNA (anticodon GAA)--polypeptide-chain 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.

Base Sequence↗

PubMed1986

Effect of replacing uridine 33 in yeast tRNAPhe on the reaction with ribosomes.

We have determined several kinetic parameters for the reaction of poly(U)-programmed ribosomes with ternary complexes of elongation factor Tu, GTP, and yeast Phe-tRNA analogs with different bases substituted for uridine in position 33. These analogs test whether disruption of the hydrogen bonds normally formed by uridine 33 and steric crowding in the anticodon loop are detrimental to tRNA function on the ribosome. Single-turnover kinetic studies of the reaction of these ternary complexes with ribosomes show that these Phe-tRNA analogs decrease the apparent rate of GTP hydrolysis (kGTP) and the ratio of peptide formed to GTP hydrolyzed. Thus, the substitution of uridine 33 affects not only the selection of a ternary complex by the ribosome but also the selection of an aminoacyl-tRNA in the proofreading reaction. The effects become greater as first one, and then the other, H-bond is disrupted. Steric crowding in the anticodon loop is also important, but does not have as great an effect on the rate constants. An analysis of the elementary rate constants which comprise the rate constant, kGTP, demonstrates that the reduction in kGTP results from a decreased rate of ternary complex association with the ribosome (k1) and that there is little or no effect on the rate of GTP cleavage (k2). An analysis of the rate constants involved in proofreading shows that all the modified (tRNAs have increased rates of aminoacyl-tRNA rejection (k4) but that the rate of peptide bond formation (k3) is unaffected.

Base Sequence↗

PubMed1986

The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis.

The fidelity of protein synthesis depends on the rate constants for the reaction of ribosomes with ternary complexes of elongation factor Tu (EF-Tu), GTP, and aminoacyl (aa)-tRNA. By measuring the rate constants for the reaction of poly(U)-programmed ribosomes with a binary complex of elongation factor (EF-Tu) and GTP we have shown that two of the key rate constants in the former reaction are determined exclusively by ribosome-EF-Tu interactions and are not affected by the aa-tRNA. These are the rate constant for GTP hydrolysis, which plays an important role in the fidelity of ternary complex selection by the ribosome, and the rate constant for EF-Tu.GDP dissociation from the ribosome, which plays an equally important role in subsequent proofreading of the aa-tRNA. We conclude that the fidelities of ternary complex selection and proofreading are fundamentally dependent on ribosome-EF-Tu interactions. These interactions determine the absolute value of the rate constants for GTP hydrolysis and EF-Tu.GDP dissociation. The ribosome then uses these rate constants as internal standards to measure, respectively, the rate constants for ternary complex and aa-tRNA dissociation from the ribosome. These rates, in turn, are highly dependent on whether the ternary complex and aa-tRNA are cognate or near-cognate to the codon being translated.

Escherichia coli↗

PubMed1986

Elongation factor Tu.guanosine 3'-diphosphate 5'-diphosphate complex increases the fidelity of proofreading in protein biosynthesis: mechanism for reducing translational errors introduced by amino acid starvation.

Complexes of elongation factor Tu (EF-Tu) with guanosine 3'-diphosphate 5'-diphosphate (ppGpp) bind to ribosomes where they slow the incorporation of aminoacyl-tRNAs into protein by inhibiting both the binding of aminoacyl-tRNA.EF-Tu.GTP ternary complexes and the formation of peptide bonds. The latter action increases the time available for aminoacyl-tRNA rejection by the ribosome and, therefore, increases the effectiveness of proofreading. Synthesis of ppGpp and the formation of EF-Tu.ppGpp occur in vivo in response to amino acid starvation. Our finding, therefore, suggests an explanation for the otherwise puzzling observation that amino acid starvation has, at most, a moderate effect on the fidelity of protein synthesis in wild-type Escherichia coli. We suggest that an EF-Tu.ppGpp-induced increase in the effectiveness of proofreading buffers the overall translational fidelity of these cells against amino acid starvation-induced errors in initial selection of aminoacyl-tRNA ternary complexes.

Escherichia coli↗