Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system

Abstract

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages.Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNAAGG in the cell to become sequestered in translating peptidyl_tRNAAGG_mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome to frameshift, hop, or terminate translation.Most amino acids are encoded by more than one codon, and frequencies of use of synonymous codons tend to reflect the relative abundance of the tRNAs that recognize them (7,29). Synonymous codons may be translated at different rates (16,17) MATERUILS AND METHODSCodon test plasmids. Plasmids pCT1, pCT2, and pCT3 ( Fig. 1) were assembled from elements of pET vectors (13,24) and of T7 DNA (6). Elements were assembled by using a natural XbaI site (T'CTAGA, where the primer indicates the position of cleavage) between the 410 promoter and the slO translation initiation region for the gene 10 major capsid protein of T7, a natural NdeI site (CA'TATG) at the slO initiation codon, and newly introduced XbaI or NheI (G'CTAGC) sites at the ends of elements. Plasmids were assembled in modular fashion by fusions of compatible ends of DNA fragments from plasmids carrying individual elements or combinations of elements. Most of the fusions were between fragments that had one end at the PstI site in the on May 7, 2018 by guest

Ribosome kinetics and aa-tRNA competition determine rate and fidelity of peptide synthesis

“…Conversely, if they increased the concentrations of cognate tRNA, then the A-site vacancy time was reduced and the frameshift frequency dropped. The aforementioned results are consistent with those of Rosenberg et al (1993), who found that mRNA containing high numbers of the rare codon AGG caused its corresponding tRNA to be sequestered and the ribosome stalled at the first of two consecutive AGG codons. The stalled ribosome frameshifted, hopped, or terminated translation as an upstream translating ribosome approached.…”

Ribosome‐mediated translational pause and protein domain organization

“…However, we propose that these two slowly translated codons are avoided by slow regions (that occur at mRNA spans that correspond to the strategic positions on proteins such as domain ends) because they may induce dissociation of the translation complex and cause a deleterious rather an intentional pause. Many reports in the literature (Robinson et al, 1984;Bonekamp et al, 1985;Spanjaard & van Duin, 1988;Varenne et al, 1989, Chen & Inouye, 1990Spanjaard et al, 1990;Kinnaird et al, 1991;Rosenberg et al, 1993;Goldman et al, 1995) show that the introduction of cua (Leu) or agg (Arg) codon strings results in the reduction of protein yield and presumably induces dissociation of the translation complex or frameshifting and hopping (Kane et al, 1992) events. Chen and Inouye (1990) observed preferential use of such codons in the region spanning the first 25 codons of the mRNA; they suggest that such a preference for the minor codons in an early gene section may modulate gene expression by premature termination of translation, thereby avoiding unnecessary translation of a large part of the mRNA.…”

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

“…We chose to study the rare Arg codons AGA and AGG [termed "AGR" according to International Union of Pure and Applied Chemistry (IUPAC) conventions] because the literature suggests that they are among the most difficult codons to replace and that their similarity to ribosome-binding sequences (RBSs) underlies important noncoding functions (8,(27)(28)(29)(30). Furthermore, their sparse use (123 instances in the essential genes of E. coli MG1655 and 4,228 instances in the entire genome) ( Table 1 and Dataset S1) made replacing all AGR instances in essential genes a tractable goal, with essential genes serving as a stringent test set for identifying any fitness impact from codon replacement (31).…”