The influence of ribosome‐binding‐site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo

Abstract

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.

High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors

“…These include a one-nucleotide exchange in the characteristic direct repeat sequences upstream of the 210 region (Brurberg et al, 1997), a one-nucleotide difference in the spacing between the putative ribosome-binding site (AGGAG in both cases) and the start codon, and sequence differences in the spacer regions. It is well known that changes in the length and/or sequence of the window between the ribosome-binding site and the start codon may affect expression Vellanoweth & Rabinowitz, 1992), and this has also been observed in an earlier variant of a P orfX -based expression system (Mathiesen et al, 2004a). …”

Pheromone cCF10 and plasmid pCF10‐encoded regulatory molecules act post‐transcriptionally to activate expression of downstream conjugation functions

“…The prgS region contains an open reading frame that could encode a 10.5 kDa basic protein, but has a relatively poor ribosome-binding site (GAGAAGN 9 GUG, whereas AGGAGGN 7-9 AUG corresponds to the most efficient ribosome-binding sites; Vellanoweth and Rabinowitz, 1992). Although the precise function of PrgS is not yet known, unregulated expression appears to be lethal to E. faecalis, expression in Escherichia coli strains radically alters colony morphology, and overexpression in E. coli leads to rapid cell lysis (B.…”

Peptide Conjugated Phosphorodiamidate Morpholino Oligomers Increase Survival of Mice Challenged with AmesBacillus anthracis

“…Targeting positions immediately upstream of the start codons of either acpP or gyrA was more effective than targeting positions downstream. A likely explanation for this effect is an overlap with the apparent ribosome binding site, which is separated 7-9 bases upstream from the start codon (Vellanoweth and Rabinowitz, 1992). Although these results show a correlation between MIC and position of the PPMO targeting upstream of the start codon, the sample size is too small to make a generalized statement.…”