Stanislas Varenne

Translation is a non-uniform process: Effect of tRNA availability on the rate of elongation of nascent polypeptide chains☆

We reported elsewhere (Varenne et al., 1982) that, during synthesis of a number of colicins in Escherichia coli, intermediate nascent chains of discrete sizes accumulated, suggesting a variable rate of translation. In this paper, a detailed analysis provides arguments that this phenomenon, at least for the proteins under study, is not related to aspects of messenger RNA such as secondary structure. It is linked to the difference in transfer RNA availability for the various codons. Experimental analysis of translation of other proteins in E. coli confirms that the main origin for the discontinuous translation in the polypeptide elongation cycle is the following. For a given codon, the stochastic search of the cognate ternary complex (aminoacyl-tRNA-EF-Tu-GTP) is the rate-limiting step in the elongation cycle: transpeptidation and translocation steps are much faster. The degree of slackening in ribosome movement is almost proportional to the inverse of tRNA concentrations. The verification of this model and its possible physiological significance are discussed.

Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate*

The complete nucleotide sequence of the structural gene for colicin A has been established. This sequence consists of 1776 base-pairs. According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989. The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured. The rest of the molecule (residues 186 to 592) is very rich in alpha-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A. A similar region has been found in colicin E1, which has the same mode of action as colicin A. Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions. The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.

Effect of distribution of unfavourable codons on the maximum rate of gene expression by an heterologous organism

We have analysed theoretically the effect of the relative position of unfavourable codons on the maximum level of synthesis of foreign proteins in E. coli. We predict that the occurrence of such codons scattered in the corresponding genes has little effect. In contrast, clustering (in our terminology indicating directly adjacent codons) of unfavourable codons is predicted to dramatically reduce the maximum level of protein synthesis. The context effect would explain the reduction of expression level for a chloramphenicol acetyl transferase gene modified by Robinson et al. (1984), which contains 4 contiguous unfavourable codons. As an example, we predict that due to the different downstream contexts of unfavourable codons in the alpha 1 and beta interferon genes, the maximum level of synthesis in E. coli for these proteins will be different.

Variable rate of polypeptide chain elongation for colicins A, E2 and E3.

Abstract Translation in vivo of colicin A messenger RNA was studied. Accumulation of nascent chains of discrete sizes was observed, providing evidence that the rate of chain elongation during colicin A biosynthesis was not uniform. This discontinuous translation was independent of induction of colicin A by mitomycin C and of the bacterial host for colicin A plasmid. A variable rate of chain elongation in vivo was also demonstrated for colicins E2 and E3. The possible roles of codon usage and mRNA secondary structure are discussed.

The maximum rate of gene expression is dependent in the downstream context of unfavourable codons

Presented here is an experimental demonstration of our theoretical predictions on the role of the downstream context of unfavourable codons in a gene on its expression level. Six non clustered AGG codons were inserted in the chloramphenicol acetyltransferase (cat) gene of E. coli and the expression of this modified gene (cat4) was compared with that of a cat gene in which four clustered AGG codons were inserted (cat2 gene). As predicted, the rate of production of the corresponding CAT4 and CAT2 proteins is equal as long as the rate of transcription of the gene does not exceed a given limit. When this limit is exceeded, production of CAT4 continues to increase, whereas CAT2 production decreases dramatically. Various consequences and possible applications of this downstream context effect are discussed.

A Mutant of Escherichia coli Deficient in Pyruvate Formate Lyase

SummaryA genetic study was performed on a mutation which has caused loss of pyruvate formate lyase. The gene affected is designated pfl and is located close to ser C, i.e. at 20 mn on the chromosomal map of E. coli.

Preferential sensitivity of syntheses of exported proteins to translation inhibitors of low polarity in Escherichia coli

SummaryWe have compared the sensitivities to different translation inhibitors of the syntheses of enzymes from various cell compartments. Alkaline and acid phosphatases were chosen as representative of periplasmic enzymes. Aminopeptidase N and β-galactosidase represented peripheral membrane protein and cytoplasmic enzyme respectively. Antibiotics of low polarity such as spectinomycin and tetracycline, when used at low concentrations, caused a preferential inhibition of exported proteins compared to total cell proteins, to aminopeptidase N and to β-galactosidase. Synthesis itself was inhibited as demonstrated by immunochemical assay of the amount of proteins synthesized in the presence or absence of spectinomycin. Temperature-dependance of the penetration of spectinomycin as reported by its effect, indicated that this drug does not enter the cell by simple dissolution in the membrane lipids but rather enters at specific sites of the cell envelope. In contrast to low polarity inhibitors, a polar antibiotic such as kanamycin did not cause any preferential inhibition of the syntheses of exported proteins. By using radioactive tetracycline (0.025, 0.1 and 0.2 μg/ml) we have investigated the distribution of this antibiotic between free and membrane-bound polysomes isolated without the use of lysozyme. Three times as much tetracycline was found in the membrane-bound compared to free polysomes fraction.Therefore we propose that preferential sensitivity of synthesis of exported proteins to translation inhibitors of low polarity reflects a preferential binding of these antibiotics to membrane-bound polysomes fractions in Escherichia coli. We thus suggest that this preferential binding occurs since the site of entry of weakly ionized antibiotics used in this study, correspond to the site of synthesis and transfer of exported proteins.