The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria

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Abstract
Internal Shine–Dalgarno-like sequences in bacterial messenger RNA determine the elongation rate of protein synthesis and synonymous codon usage. The rate of protein synthesis by the ribosome can be influenced by various intrinsic and extrinsic factors, such as structure in the messenger RNA and the actions of RNA binding proteins. As a result, translation is subject to transient pausing. This study provides a genome-wide view of the locations of ribosome pausing in bacteria. The most common origin of pausing is found to be hybridization between the ribosomal RNA and internal sequences of the mRNA that are similar to the Shine–Dalgarno element that serves as the entry point for the ribosome. To prevent their interference with translation, such Shine–Dalgarno-like elements are underrepresented in protein-coding sequences, accounting for some of the observed bias in codon usage. These results have implications both for our basic understanding of protein synthesis and for practical efforts to express recombinant proteins. Protein synthesis by ribosomes takes place on a linear substrate but at non-uniform speeds. Transient pausing of ribosomes can affect a variety of co-translational processes, including protein targeting and folding1. These pauses are influenced by the sequence of the messenger RNA2. Thus, redundancy in the genetic code allows the same protein to be translated at different rates. However, our knowledge of both the position and the mechanism of translational pausing in vivo is highly limited. Here we present a genome-wide analysis of translational pausing in bacteria by ribosome profiling—deep sequencing of ribosome-protected mRNA fragments3,4,5. This approach enables the high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare transfer RNAs do not lead to slow translation under nutrient-rich conditions. Instead, Shine–Dalgarno-(SD)6-like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome7,8 possessing an altered anti-SD sequence, we show that pausing is due to hybridization between the mRNA and 16S ribosomal RNA of the translating ribosome. In protein-coding sequences, internal SD sequences are disfavoured, which leads to biased usage, avoiding codons and codon pairs that resemble canonical SD sites. Our results indicate that internal SD-like sequences are a major determinant of translation rates and a global driving force for the coding of bacterial genomes.