Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro

Abstract

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNA f . All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E. coli. Addition of large amounts of E. coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause-site peptides seen in the expression of wild-type CAT. Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

A Synonymous Single Nucleotide Polymorphism in ΔF508 CFTR Alters the Secondary Structure of the mRNA and the Expression of the Mutant Protein

“…Therefore, it is unlikely that the slight reduction of the codon frequency in a single locus (47% (ATC) to 35% (ATT)) with relative synonymous codon usage values changing from 20.9 to 15.8 would significantly affect tRNA availability. A study investigating the role of SNPs on CAT synthesis supports this idea (57). Because excess amounts of Escherichia coli tRNAs did not alter the pause cycles, the authors suggested that mRNA structural alterations, rather than the limited availability of rare tRNAs, were responsible for translational pausing.…”

“…Although our studies concentrated on the mRNA secondary structural alterations caused by the deletion of CTT in the human CFTR and introduction of the synonymous codon (ATT) for Ile-507, we also analyzed the codon frequency for Ile in the human genome and in CFTR. We did this based on previous studies suggesting that clusters of rare codons may cause translational pauses (55)(56)(57)(58). We found that that while ATT is a less frequent codon than ATC, ATA is the rarest codon for Ile in the human genome.…”

“…WT and ⌬F508 CFTR mRNA Half-life Measurements-CFTR mRNA half-lives were measured in HeLaWT or HeLa⌬F cells (33) cultured under standard conditions as described (57). The mRNA half-lives were calculated from the exponential decay based on: C/C 0 ϭ e Ϫkdt (C and C 0 are mRNA amounts after time t and at t 0 , respectively, and k d is the mRNA decay constant).…”

Molecular simulations of cotranslational protein folding: fragment stabilities, folding cooperativity and trapping in the ribosome tunnel

“…That said, it should also be noted that this matching has been done assuming that amino acid synthesis occurs at a constant rate; in reality, however, there can be significant variations (up to 25-fold) in translation rates, depending on the availability of the appropriate cognate tRNAs (e.g., [63]), the nature of the base-pairing interaction between codon and anticodon (e.g., [64,65]), and/or the presence of secondary structure in the coding mRNA (e.g., [66]). There has been some discussion in the literature that slowly translated regions may, in general, occur at boundaries between protein domains (e.g., [67,68]), and this issue could be investigated in future for proteins in which the effects of different codons on translational pausing have been studied (e.g., [69]). For the more limited goals of the present work, however, the comparison shown in Figure 11A suggests that the choice of a (constant) simulated rate of one amino acid per 100 ns at least provides a reasonable compromise between the need to allow the nascent chain to sample a wide range of conformations during synthesis and the need to make the simulations sufficiently rapid that many independent trajectories, performed under many different simulation conditions, can be obtained.…”

Experimental and Computational Analysis of Translation Products in Apomyoglobin Expression

“…87 Finally, only 20% of the detected ribosomes were regarded as active, according to a typical assessment in E. coli cell-free systems. 88 …”