An Improved Caulobacter crescentus Operon Annotation Based on Transcriptome Data

Bharmal, Mohammed-Husain M.; Aretakis, James R.; Schrader, Jared M.

doi:10.1128/mra.01025-20

Cited by 13 publications

(16 citation statements)

References 7 publications

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“…As expected, we observed significant peak within the hfiA promoter region (Figure 4A). Peaks were highly enriched near globally defined transcription start sites (TSS) [30-32] when compared to a set of randomly generated peaks (Figure 4B); this TSS-proximal enrichment pattern is characteristic of proteins that directly bind DNA to regulate gene expression. To identify putative binding motifs in the ChIP-seq peaks, we analyzed the peak sequences using the XSTREME algorithm within the MEME Suite [33].…”

Section: Resultsmentioning

confidence: 99%

“…The Bioconductor package was used to identify overlap between RtrC-regulated genes defined by RNA-seq and RtrC binding sites defined by ChIP-seq [61]. Promoters for genes were designated as the sequence -400 to +100 around the TSS [30]. Overlap between promoters and RtrC motifs identified from XSTREME was analyzed using ChIPpeakAnno within Bioconductor.…”

Section: Rna-seq and Chip-seq Overlap Analysismentioning

confidence: 99%

“…By combining RNA-seq and ChIP-seq datasets, we identified genes that are directly controlled by RtrC. Direct targets were defined as genes that a) were differentially regulated in rtrC ++ relative to an empty vector control, b) contained an RtrC-enriched peak by ChIP-seq, and c) contained an RtrC binding motif in their promoter region [32]. Of the directly regulated genes, 63% were activated and 37% were repressed by RtrC (Figure 5A; Table S3).…”

Section: Rtrc Is a Transcriptional Activator And Repressormentioning

confidence: 99%

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A cryptic transcription factor regulates Caulobacter adhesin development

McLaughlin

Hershey

Reyes-Ruiz

et al. 2022

Preprint

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Alphaproteobacteria commonly produce an adhesin that is anchored to the exterior of the envelope at one cell pole. In Caulobacter crescentus, this adhesin enables permanent attachment to solid surfaces and is known as the holdfast. An ensemble of two-component signal transduction (TCS) proteins control C. crescentus holdfast biogenesis by indirectly regulating expression of HfiA, a potent inhibitor of holdfast synthesis. A genetic selection to discover direct hfiA regulators that function downstream of this adhesion TCS system identified a hypothetical gene that we have named rtrC. Though the primary structure of RtrC bears no resemblance to any defined protein family, RtrC directly binds and regulates dozens of sites on the C. crescentus chromosome via a pseudo-palindromic motif. Among these binding sites is the hfiA promoter, where RtrC functions to directly repress transcription and thereby activate holdfast development. RtrC, the DNA-binding response regulator SpdR, and the transcription factor RtrB together form an OR-gated type I coherent feedforward loop (C1-FFL) that regulates hfiA transcription. C1-FFL motifs are known to buffer gene expression against transient loss of regulating signals, which often occurs in fluctuating natural environments. We conclude that the formerly hypothetical gene, rtrC, encodes a transcription factor that functions downstream of the C. crescentus TCS adhesion control system to regulate development of the holdfast adhesin.

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Section: Resultsmentioning

confidence: 99%

Section: Rna-seq and Chip-seq Overlap Analysismentioning

confidence: 99%

Section: Rtrc Is a Transcriptional Activator And Repressormentioning

confidence: 99%

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A cryptic transcription factor regulates Caulobacter adhesin development

McLaughlin

Hershey

Reyes-Ruiz

et al. 2022

Preprint

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“…Bacterial transcriptome architecture maps provide an ideal platform to analyze TIR accessibility on a transcriptome-wide scale. Notably, transcription start site (TSS) data and RNA-seq density data have been used previously to experimentally map bacterial transcriptomes (33). If such TSS data are available, the user can input this together with the genbank file of the respective genome containing the CDS annotations and an operon map to build a transcript architecture model that allows the best starting point for transcriptome analyses.…”

Section: Methodsmentioning

confidence: 99%

ΔG_unfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization

Bharmal

Schrader

2021

Preprint

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Translation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). ΔGunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput. ΔGunfold leaderless allows users to automate the ΔGunfold calculation to perform high-throughput analysis. Importantly, ΔGunfold leaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5' UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9). The ΔGunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.

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“…Further genome wide analysis revealed that SD sites are also prevalent within the coding sequence where they may induce ribosome pausing and are predicted to be non-functional for initiation (8)(9)(10), although neither has been thoroughly tested. The bacterium C. crescentus is the fastest growing species that is predominantly nonSD in translation initiation and it also has a well annotated transcriptome (11,12), making it an ideal model to probe how mRNA sequence features program translation initiation in TIRs as opposed to AUGs in the CDS (called elongator AUG regions henceforth) in predominantly nonSD species.…”

Section: Introductionmentioning

confidence: 99%

Initiator AUGs are discriminated from elongator AUGs predominantly through mRNA accessibility inC. crescentus

Ghosh

Bharmal

Ghaleb

et al. 2022

Preprint

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Translation initiation in bacteria is thought to occur upon base-pairing between the Shine-Dalgarno site in the mRNA and anti-Shine-Dalgarno site in the rRNA. However, in many bacterial species, such as Caulobacter crescentus, a minority of mRNAs have Shine-Dalgarno sites. To examine the functional importance of Shine-Dalgarno sites in C. crescentus, we analyzed the transcriptome and found more Shine-Dalgarno sites exist in the coding sequence than preceding start codons. To examine the function of Shine-Dalgarno sites in initiation we designed a series of mutants with altered ribosome accessibility and Shine-Dalgarno content in translation initiation regions (TIRs) and elongator AUG regions (EARs). A lack of mRNA structure content is required for initiation in TIRs, and when introduced into EARs, can stimulate initiation, suggesting that low mRNA structure content is a major feature required for initiation. SD sites appear to stimulate initiation in TIRs, which generally lack structure content, but SD sites only stimulate initiation in EARs if RNA secondary structures are destabilized. Taken together, this suggests that the difference in secondary structure between TIRs and EARs directs ribosomes to start codons where SD base pairing can tune the efficiency of initiation, but SDs in EARs do not stimulate initiation as they are blocked by stable secondary structures. This highlights the importance of studying translation initiation mechanisms in diverse bacterial species.

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An Improved Caulobacter crescentus Operon Annotation Based on Transcriptome Data

Cited by 13 publications

References 7 publications

A cryptic transcription factor regulates Caulobacter adhesin development

A cryptic transcription factor regulates Caulobacter adhesin development

ΔG_unfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization

Initiator AUGs are discriminated from elongator AUGs predominantly through mRNA accessibility inC. crescentus

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An Improved Caulobacter crescentus Operon Annotation Based on Transcriptome Data

Cited by 13 publications

References 7 publications

A cryptic transcription factor regulates Caulobacter adhesin development

A cryptic transcription factor regulates Caulobacter adhesin development

ΔGunfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization

Initiator AUGs are discriminated from elongator AUGs predominantly through mRNA accessibility inC. crescentus

Contact Info

Product

Resources

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ΔG_unfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization