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High-resolution transcriptome and genome-wide dynamics of RNA polymerase and NusA in Mycobacterium tuberculosis (RNA-seq)

ABSTRACT: To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches, strand-specific RNA-seq and ChIP-seq, to survey the global transcriptome and monitor genome-wide dynamics of RNA polymerase (RNAP) and NusA. ChIP-seq revealed that RNAP and the antiterminator, NusA, occurred ubiquitously with the NusA profile mirroring RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the datasets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align.

ORGANISM(S): Mycobacterium tuberculosis

PROVIDER: GSE40846 | GEO | 2012/12/01

SECONDARY ACCESSION(S): PRJNA175135

REPOSITORIES: GEO

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High-resolution transcriptome and genome-wide dynamics of RNA polymerase and NusA in Mycobacterium tuberculosis (RNA-seq)

Project description:To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches, strand-specific RNA-seq and ChIP-seq, to survey the global transcriptome and monitor genome-wide dynamics of RNA polymerase (RNAP) and NusA. ChIP-seq revealed that RNAP and the antiterminator, NusA, occurred ubiquitously with the NusA profile mirroring RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the datasets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align. RNA-Seq in exponential and stationary phase cultures

2012-12-01 | E-GEOD-40846 | biostudies-arrayexpress

High-resolution transcriptome and genome-wide dynamics of RNA polymerase and NusA in Mycobacterium tuberculosis (ChIP-seq)

Project description:To construct a regulatory map of the genome of the human pathogen, Mycobacterium tuberculosis, we applied two complementary high-resolution approaches, strand-specific RNA-seq and ChIP-seq, to survey the global transcriptome and monitor genome-wide dynamics of RNA polymerase (RNAP) and NusA. ChIP-seq revealed that RNAP and the antiterminator, NusA, occurred ubiquitously with the NusA profile mirroring RNAP distribution in both the exponential and stationary phases of growth. Generally, promoter-proximal peaks for RNAP and NusA were observed, followed by a decrease in signal strength reflecting transcriptional polarity. Differential binding of RNAP and NusA in the two growth conditions correlated with transcriptional activity as reflected by RNA abundance. Indeed, a significant association between expression levels and the presence of NusA throughout the gene body was detected, confirming the peculiar transcription-promoting role of NusA. Integration of the datasets pinpointed transcriptional units, mapped promoters and uncovered new anti-sense and non-coding transcripts. Highly expressed transcriptional units were situated mainly on the leading strand, despite the relatively unbiased distribution of genes throughout the genome, thus helping the replicative and transcriptional complexes to align. ChIP-Seq of RNAP and NusA in Mtb H37Rv at exponential and stationary phase cultures. Each experiment was performed in duplicate. Input DNA (No IP) was used as a control.

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Roles for the Transcription Elongation Factor NusA in Both DNA Repair and Damage Tolerance Pathways in Escherichia coli

Project description:Here we report our observations that have led us to propose that the transcription elongation factor NusA promotes a novel class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier we have reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to ultraviolet radiation. Gene expression profiling suggests this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an N2-furfuryl-dG (N2-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the non-transcribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in a novel class of TCR involved in the identification and removal of a class of lesion, such as the N2-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.

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Project description:In this work we conducted RNET-seq on Wild Type, NusA depletion, nusG deletion, and NusA depletion nusG deletion strains of B. subtilis. Using this data we analysed the transcriptome-wide effects of NusA on RNA polymerase pausing and found that NusA has modest pause stimulating as well as suppressing effects throughout the genome.

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Project description:In all organisms, the multi-subunit RNA polymerases (RNAPs) that synthesize messenger RNAs bind multiple accessory proteins to regulate transcript elongation rate, transcriptional pausing, and termination. However, the dynamics of regulatory protein association/dissociation and how different regulators influence one another’s function is unclear. We used single-molecule multi-wavelength fluorescence colocalization techniques to directly observe the dynamics of the elongation regulators NusA and σ70 with E. coli RNAP in vitro. Contrary to previous proposals, NusA was observed to repeatedly bind to and release from elongation complexes (ECs) during synthesis of a single RNA. However, elongation complexes that retained bound σ70 did not bind NusA and the EC-bound σ70 was largely retained even in the presence of physiological (µM) concentrations of competing elongation factors NusA and/or NusG. Factor occupancy of elongation complexes was non-equilibrium, with substantial amounts of σ70ECs even in the absence of free σ70, because composition was controlled by slow σ70 dissociation from σ70ECs. The experiments show that at steady state the same gene is transcribed by two distinct types of elongation complexes that are (ECs) or are not (σ70ECs) capable of binding NusA. Consistent with the known regulatory effects of NusA, the two types are observed to have different elongation rates, pausing dynamics, and efficiency of termination at an intrinsic terminator. During cellular transcription, these non-equilibrium populations of ECs are predicted to cause selective EC loss at intergenic transcriptional attenuators, traffic jams and altered pausing. This is an example of a potentially general “two-body” mechanism in which a functionally silent protein fine-tunes gene expression by modulating a second, functionally consequential regulator.

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Regulator trafficking on bacterial transcription units in vivo

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Disributions of RpoC SigA NusA and GreA in Bacillus subtilis

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