Ribosome profiling reveals conserved guanosine residues at ribosome pausing sites in Bacillus subtilis

Ribosome pausing slows down translation and can affect protein synthesis. Improving translation efficiency can therefore be of commercial value. Here, we investigated the occurrence of ribosome pausing during amylase secretion by the industrial production organism Bacillus subtilis under repeated batch fermentation conditions. Interestingly, we found an enrichment of guanosine residues in the first nucleotide of the E and P site at ribosome pause sites, and in the first nucleotide of the fourth codon upstream of the ribosome pause site. This sequence motif deviates from the earlier described enrichment of Shine-Dalgarno-like sequences or glycine codons at ribosome pause sites. For the highly expressed amylase gene amyM several strong ribosome pause sites were detected, which remained for the most part present during the 64-hour long fermentation, despite changes in nutrient availability. These pause sites were neither related to rare codons nor to secondary protein structures, suggesting that secondary mRNA structures might be critical for ribosome pausing. When surveying the genome, an interesting finding was the presence of strong ribosome pause sites in several toxins genes. These potential ribosome stall sites may function in preventing inadvertent activity in the cytosol by means of delaying full translation.