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

ABSTRACT 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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    • "Note that this special case agrees well with the results described in [23] (see (1)). It is important to emphasize, however, that there are various possible intracellular mechanisms that may affect λ j i , i > 0. For example, synonymous mutation/changes (in endogenous or heterologous) genes inside the coding region may affect the adaptation of codons to the tRNA pool (codons that are recognized by tRNA with higher intracellular abundance usually tend to be translated more quickly [15]), the local folding of the mRNA (stronger folding tend to decrease elongation rate [65]), or the interaction/hybridization between the ribosomal RNA and the mRNA [34] (there are nucleotides sub-sequence that tend to interact with the ribosomal RNA, causing transient pausing of the ribosome, and delay the translation elongation rate). Non synonymous mutation/changes inside the coding region may also affect the elongation for example via the interaction between the nascent peptide and the exit tunnel of the ribosome [37], [55]. "
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    ABSTRACT: Large-scale simultaneous mRNA translation and the resulting competition for the available ribosomes has important implications to the cell's functioning and evolution. Developing a better understanding of the intricate correlations between these simultaneous processes, rather than focusing on the translation of a single isolated transcript, should help in gaining a better understanding of mRNA translation regulation and the way elongation rates affect organismal fitness. A model of simultaneous translation is specifically important when dealing with highly expressed genes, as these consume more resources. In addition, such a model can lead to more accurate predictions that are needed in the interconnection of translational modules in synthetic biology. We develop and analyze a general model for large-scale simultaneous mRNA translation and competition for ribosomes. This is based on combining several ribosome flow models (RFMs) interconnected via a pool of free ribosomes. We prove that the compound system always converges to a steady-state and that it always entrains or phase locks to periodically time-varying transition rates in any of the mRNA molecules. We use this model to explore the interactions between the various mRNA molecules and ribosomes at steady-state. We show that increasing the length of an mRNA molecule decreases the production rate of all the mRNAs. Increasing any of the codon translation rates in a specific mRNA molecule yields a local effect: an increase in the translation rate of this mRNA, and also a global effect: the translation rates in the other mRNA molecules all increase or all decrease. These results suggest that the effect of codon decoding rates of endogenous and heterologous mRNAs on protein production is more complicated than previously thought.
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    • "MNase yields a broad distribution of fragment lengths, none of which are superior at conveying information about position or reading frame. Given the apparent variation at both ends of the RNA fragments, in early bacterial profiling studies, ribosome occupancy was broadly distributed across the center of the reads and not assigned specifically to the 5 0 or 3 0 end (Li et al., 2012; Oh et al., 2011). This has the undesirable effect of blurring the signal. "
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    ABSTRACT: Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here, we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream, and we identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp-knockout strain. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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