Translational initiation in E. coli occurs at the correct sites genome-wide in the absence of mRNA-rRNA base-pairing
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ABSTRACT:
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
PROVIDER: GSE135906 | GEO | 2020/02/21
REPOSITORIES: GEO
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Project description:Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. We provide a universal strategy for defining the coding potential of bacterial genomes by applying ribosome profiling, RNA-seq, global 5’-RACE, and liquid chromatography coupled with tandem mass spectrometry (LC-MS) data to the 4-megabase C. crescentus genome. We mapped transcript units at single base-pair resolution using RNA-seq together with global 5’ RACE. Additionally, using ribosome profiling and LC-MS, we mapped translation start sites and coding regions with near complete coverage. We found most start codons lacked corresponding Shine-Dalgarno sites although ribosomes were observed to pause at internal Shine-Dalgarno sites within the ORF. These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. Overall 19% of the transcribed and translated genomic elements were newly identified or significantly improved by this approach providing a valuable genomic resource to elucidate the complete C. crescentus genetic circuitry that controls asymmetric cell division. Ribosome profiling and RNA-seq data were collected in Caulobacter crescentus NA1000 cells grown in M2G and PYE media to map transcript and ORF features in the genome.
2014-05-29 | E-GEOD-54883 | biostudies-arrayexpress
Project description:Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. We provide a universal strategy for defining the coding potential of bacterial genomes by applying ribosome profiling, RNA-seq, global 5’-RACE, and liquid chromatography coupled with tandem mass spectrometry (LC-MS) data to the 4-megabase C. crescentus genome. We mapped transcript units at single base-pair resolution using RNA-seq together with global 5’ RACE. Additionally, using ribosome profiling and LC-MS, we mapped translation start sites and coding regions with near complete coverage. We found most start codons lacked corresponding Shine-Dalgarno sites although ribosomes were observed to pause at internal Shine-Dalgarno sites within the ORF. These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. Overall 19% of the transcribed and translated genomic elements were newly identified or significantly improved by this approach providing a valuable genomic resource to elucidate the complete C. crescentus genetic circuitry that controls asymmetric cell division.
2014-05-29 | GSE54883 | GEO
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
2012-04-01 | E-GEOD-35641 | biostudies-arrayexpress
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.
2012-04-01 | GSE35641 | GEO
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
Project description:Ribosomes of Bacteroidia (formerly Bacteroidetes) fail to recognize Shine-Dalgarno (SD) sequences even though they harbor the anti-SD (ASD) of 16S rRNA. This is due to sequestration of the 3’ tail of 16S rRNA in a pocket formed by bS21, bS18, and bS6 on the 30S platform, which functionally occludes the ASD. Interestingly, in many Flavobacteriales, the gene encoding bS21, rpsU, contains an extended SD sequence. In this work, we present genetic and biochemical evidence that bS21 synthesis in Flavobacterium johnsoniae is autoregulated via a subpopulation of ribosomes that specifically lack bS21. Mutation or depletion of bS21 in the cell specifically increases translation of reporters with strong SD sequences, such as rpsU’-gfp. Purified ribosomes lacking bS21 (or its C-terminal region) exhibit higher rates of initiation on rpsU mRNA and lower rates of initiation on other (SD-less) mRNAs than control ribosomes. The mechanism of autoregulation depends on extensive pairing between mRNA and 16S rRNA, and exceptionally strong SD sequences, with predicted pairing free energies of < –13 kcal/mol, are characteristic of rpsU across the Bacteroidia. To our knowledge, this is the first example of biochemically-verified specialized ribosomes with a clear regulatory purpose.
2023-03-11 | PXD036353 | Pride
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.
2016-01-13 | GSE72899 | GEO
Project description:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.
2019-06-05 | GSE125086 | GEO
Project description:The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The textbook knowledge of eukaryotic translation initiation depicts 5’→3’ unidirectional migration of the pre-initiation complex (PIC) along the 5’UTR. In probing translation initiation from ultra-short 5’UTR, we report that an AUG triplet near the 5’ end can be selected via PIC backsliding. The bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5’ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5’ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.
2021-11-24 | GSE176058 | GEO
Project description:eIF3 is a multi-subunit complex thought to execute numerous functions in canonical translation initiation, including mRNA recruitment to the 40S ribosome, scanning for the start codon, and inhibition of 60S subunit joining 1–3. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors 4, but a direct involvement in translation elongation has never been demonstrated. Using selective ribosome profiling, we made the unexpected observation that eIF3 remains bound to post-initiation 80S ribosomes, followed by release after translation of ~50 codons. Furthermore, eIF3 deficiency reduces early ribosomal elongation speed, particularly on mRNAs encoding proteins associated with membrane-associated functions, resulting in defective synthesis of their encoded proteins and abnormal mitochondrial and lysosomal physiology. Accordingly, heterozygous eIF3e+/- knockout mice accumulate giant mitochondria in skeletal muscle and show a progressive decline in muscle strength with age. Hence, in addition to its canonical role in translation initiation, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for normal muscle health.
2020-06-15 | GSE149697 | GEO