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Clarifying the translational pausing landscape in bacteria by ribosome profiling

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.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE72899 | GEO | 2016/01/13

SECONDARY ACCESSION(S): PRJNA295296

REPOSITORIES: GEO

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Clarifying the translational pausing landscape in bacteria by ribosome profiling

Project description: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. Ribosome profiling (three replicates) and RNAseq (two replicates) of E. coli MG1655

2016-01-13 | E-GEOD-72899 | biostudies-arrayexpress

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Project description:Protein synthesis by ribosomes takes place on a linear substrate but at variable speeds. Transient pausing of ribosomes can impact a variety of co-translational processes, including protein targeting and folding. These pauses are influenced by the sequence of the mRNA. 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 using ribosome profiling-deep sequencing of ribosome-protected mRNA fragments. This approach enables high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare tRNAs 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 demonstrated that pausing is due to hybridization between mRNA and the 16S rRNA 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. Identification of translation pause sites in vivo using ribosome profiling

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High Precision Analysis of Translational Pausing in Bacteria Lacking EFP by Ribosome Profiling

Project description: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 identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp knockout strain. Ribosome profiling of E. coli MG1655 and mutants lacking EFP or its three modifiying enzymes

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Translational initiation in E. coli occurs at the correct sites genome-wide in the absence of mRNA-rRNA base-pairing

Project description:Shine-Dalgarno (SD) motifs are thought to play an important role in translational initiation in bacteria. Paradoxically, ribosome profiling studies in E. coli show no correlation between the strength of the SD in an mRNA and how efficiently it is translated. Performing profiling on ribosomes with altered anti-Shine-Dalgarno sequences, we reveal a genome-wide correlation between SD strength and ribosome occupancy that was previously masked by other contributing factors. Using the antibiotic retapamulin to trap initiation complexes at start codons, we find that the mutant ribosomes select start sites correctly, arguing that start sites are hard-wired for initiation through the action of other mRNA features. We show that A-rich sequences upstream of start codons promote initiation. Taken together, our genome-wide study reveals that SD motifs are not necessary for the ribosome to select where initiation occurs, though they do affect how efficiently initiation occurs at sites whose other features support initiation.

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Project description:The regulation of translation elongation plays a vital role in protein folding; an adequate translational pause provides time and cellular environments for the co-translational folding of nascent peptides. However, the genomic landscape, sequence determinants, and molecular consequences of translational pausing remain mostly unknown. In this study, we performed disome-seq that sequenced mRNA fragments protected by two consecutive ribosomes – a product of severe translational pauses during which the upstream ribosome collides into the paused one. We detected severe translational pauses on ~75% of yeast genes. These pauses were often explained by one of the three mechanisms: 1) slow ribosome releasing at stop codons, 2) slow peptide formation from proline, glycine, asparagine, and cysteine, and 3) slow leaving of polylysine from the exit tunnel of ribosomes. Notably, these amino acids also terminate the α-helical conformation. Such dual roles of amino acids establish an inborn coupling between the synthetic completion of a structural motif and a translational pause. Furthermore, paused ribosomes often recruit chaperones to assist protein folding. As a consequence, emergent protein structures during evolution should be ready to be correctly folded. Collectively, our study shows widespread translational pauses and sheds lights on a better understanding of the regulation of co-translational protein folding.

2020-12-10 | PXD015271 | Pride

Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5’UTR and Responsive to mTORC1 Signaling (Microarray)

Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Untreated TSC2 WT MEFs, TSC2 KO MEFs and TSC2 WT MEFs, TSC2 KO MEFs treated with 20nM rapamycin for 30 minutes or 3hours were harvested for ribosme profiling. The fraction samples were pooled into three groups based on velocity sedimentation: single ribosome fraction (Small group), fractions with 2 ~ 4 ribosomes (Medium group), and the one with ≥5 ribosomes (Large group). RNA were extracted from the whole cell lysis and each fraction group.

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Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5’UTR and Responsive to mTORC1 Signaling (Microarray)

Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway.

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