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


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.

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Figure 1: Analysis of translational pausing using ribosome profiling in bacteria.
Figure 2: Relationship between ribosome pausing and internal Shine–Dalgarno sequences.
Figure 3: Pausing of elongating ribosomes due to SD–aSD interaction.
Figure 4: Selection against SD-like sequences and the constraint on protein coding.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The footprint sequencing data are deposited in the Gene Expression Omnibus (GEO) under accession number GSE35641.


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We thank E. Reuman, D. Burkhardt, C. Jan, C. Gross, J. Elf and members of the Weissman laboratory for discussions; J. Dunn for ribosome profiling data on S. cerevisiae; C. Chu for help with sequencing; and J. Chin for orthogonal ribosome reagents and advice. This research was supported by the Helen Hay Whitney Foundation (to G.W.L.) and by the Howard Hughes Medical Institute (to J.S.W.).

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G.W.L. and J.S.W. designed the experiments. G.W.L. performed experiments and analysed the data. E.O. provided technical support and preliminary data. G.W.L. and J.S.W. wrote the manuscript.

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Correspondence to Jonathan S. Weissman.

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The authors declare no competing financial interests.

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Li, GW., Oh, E. & Weissman, J. The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria. Nature 484, 538–541 (2012).

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