The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria. Nature (Lond)

Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, California 94158, USA.
Nature (Impact Factor: 41.46). 03/2012; 484(7395):538-41. DOI: 10.1038/nature10965
Source: PubMed


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 folding. These pauses are influenced by the sequence of the messenger RNA. 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 fragments. 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)-like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome 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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    • "Their data exhibit a robust peak at À22 in the cross-correlation plots and slopes of 0.21–0.25, nearly as high asLi et al. (2012). These findings further support the conclusion that the correlation between aSD affinity and ribosome density strongly depends on the length of the mRNA fragments isolated. "
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    ABSTRACT: The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling.
    Full-text · Article · Jan 2016 · Cell Reports
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    • "Transcription of both the orthogonal rRNA and mRNA is required for gene expression, creating AND logic for greater control over the output [119]; the orthogonal rRNA could be considered a trans-activating RNA in this particular context. A practical consequence of the use of orthogonal rRNAs is that the coding sequence of target mRNAs may need to be reconfigured to prevent pausing at Shine–Dalgarno-like sequences [120]. "
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    ABSTRACT: Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks which require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and describe efficient strategies for rapidly identifying and correcting causes of failure and fine tuning circuit characteristics.
    Full-text · Article · Oct 2015 · Journal of Molecular Biology
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    • "Thus, it has limited applications and cannot be used for determining the reading frame (Woolstenhulme et al., 2015). Both, calibrated and center-weighted ribosomal reads can be used to assess ribosomal enrichment over specific codons (Li et al., 2012; Ishimura et al., 2014) or to determine sequences over which ribosomes transiently pause (Li et al., 2012; Woolstenhulme et al., 2015). "
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    ABSTRACT: Ribosome profiling is a new emerging technology that uses massively parallel amplification of ribosome-protected fragments and next-generation sequencing to monitor translation in vivo with codon resolution. Studies using this approach provide insightful views on the regulation of translation on a global cell-wide level. In this review, we compare different experimental set-ups and current protocols for sequencing data analysis. Specifically, we review the pitfalls at some experimental steps and highlight the importance of standardized protocol for sample preparation and data processing pipeline, at least for mapping and normalization.
    Full-text · Article · Sep 2015 · Biological Chemistry
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