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The coding and noncoding architecture of the Caulobacter crescentus genome

ABSTRACT: 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.

ORGANISM(S): Caulobacter vibrioides NA1000

PROVIDER: GSE54883 | GEO | 2014/05/29

SECONDARY ACCESSION(S): PRJNA237963

REPOSITORIES: GEO

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The coding and noncoding architecture of the Caulobacter crescentus genome

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

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.

2020-02-21 | GSE135906 | GEO

Comparative analysis of regulatory elements between Escherichia coli and Klebsiella pneumoniae by genome-wide transcription start site profiling [TSS-Seq]

Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of transcription start sites for two enterobacteria: Escherichia coli and Klebsiella pneumoniae.By obtaining over fourteen billion bases of sequence from 5' RACE (rapid amplification of cDNA ends) followed by deep sequencing, we generated genome-wide TSS (transcription start site) maps for those two species. With experimentally derived TSS datasets, we examined usage of multiple TSSs, length of 5' UTR (untranslated region), SD (Shine-Dalgarno) sequence motif, promoter sequence motif, and dinucleotide preference near TSS site. In addition, we used the TSS datasets to identify sRNAs (small RNAs) in E. coli and K. pneumoniae. Based on these analysis, we compared regulatory elements including promoter, 5' UTR and sRNAs between two species, and investigated similarities and differences of upstream regulatory regions. Moreover, sRNAs were also compared and analyzed in terms of their sequences and target sequences, presenting possible working mechanisms of K. pneumoniae sRNAs by transferring prior knowledge from E. coli sRNAs. Examination of transcription start sites by biological duplicates from E. coli and K. pneumoniae

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The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria

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

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

Comparative analysis of regulatory elements between Escherichia coli and Klebsiella pneumoniae by genome-wide transcription start site profiling [TSS-Seq]

2012-07-30 | GSE35821 | GEO

Clarifying the translational pausing landscape in bacteria by ribosome profiling

2016-01-13 | GSE72899 | GEO

Proteomics_LC-MS_MS

Project description:Proteomics LC-MS MS dataset

| EGAD00010001006 | EGA

The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria

2012-04-01 | GSE35641 | GEO

Pervasive Translation in Mycobacterium tuberculosis

Project description:The vast majority of bacterial open reading frames (ORFs) are identified by automated prediction algorithms. However, these algorithms fail to identify ORFs that deviate from the canonical features of ORFs such as a length of >50 codons, and the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to experimentally identify actively translated ORFs in Mycobacterium tuberculosis. Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. Moreover, the newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. The codon usage of the newly discovered ORFs suggests that most are not undergoing purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify ~90 new ORFs, with a median length of 52 codons, that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs serves as a rich source for the evolution of new functional proteins

2021-06-30 | E-MTAB-10695 | biostudies-arrayexpress

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