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.

1 Follower
77 Reads
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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.
    Journal of Molecular Biology 10/2015; DOI:10.1016/j.jmb.2015.10.004 · 4.33 Impact Factor
    • "From this perspective, synonymous codon swaps should be unlikely to be deleterious [13] [39]. However, while most synonymous mutations are well-tolerated [13] [39], disrupting overlapping sequence features such as ribosome binding motifs [152] and small RNAs [153] can impact crucial cellular functions. Additionally, synonymous mutations are sometimes unpredictably rejected even when non-synonymous mutations are not [39], perhaps due to mRNA structure [154] [155] or codon usage preferences [155] [156]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code.
    Journal of Molecular Biology 09/2015; DOI:10.1016/j.jmb.2015.09.003 · 4.33 Impact Factor
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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.
Show more

Preview (2 Sources)

77 Reads
Available from