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Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli.

ABSTRACT: An essential part of gene expression is the coordination of RNA synthesis and degradation, which occurs in the same cellular compartment in bacteria. Here, we report a genome-wide RNA degradation study in Escherichia coli using RNA-seq, and present evidence that the stereotypical exponential RNA decay curve obtained using initiation inhibitor, rifampicin, consists of two phases: residual RNA synthesis, a delay in the interruption of steady state that is dependent on distance relative to the mRNA's 5' end, and the exponential decay. This gives a more accurate RNA lifetime and RNA polymerase elongation rate simultaneously genome-wide. Transcripts typically have a single RNA decay constant along all positions, which is distinct between different operons, indicating that RNA stability is unlikely determined by local sequences. These measurements allowed us to establish a model for RNA processing involving co-transcriptional degradation, providing quantitative description of the macromolecular coordination in gene expression in bacteria on a system-wide level.

SUBMITTER: Chen H

PROVIDER: S-EPMC4332155 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli.

Chen Huiyi H Shiroguchi Katsuyuki K Ge Hao H Xie Xiaoliang Sunney XS

Molecular systems biology 20150112 1

An essential part of gene expression is the coordination of RNA synthesis and degradation, which occurs in the same cellular compartment in bacteria. Here, we report a genome-wide RNA degradation study in Escherichia coli using RNA-seq, and present evidence that the stereotypical exponential RNA decay curve obtained using initiation inhibitor, rifampicin, consists of two phases: residual RNA synthesis, a delay in the interruption of steady state that is dependent on distance relative to the mRNA ...[more]

PMID: 25583150

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Project description:Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH-ops interactions establish long-lived RfaH-EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS-binding features, explaining minor-paralog targeting. IMPORTANCE Bacterial pathogens adapt to hosts and host defenses by reprogramming gene expression, including by H-NS counter-silencing. Counter-silencing turns on transcription initiation when regulators bind to promoters and rearrange repressive H-NS nucleoprotein filaments that ordinarily block transcription. The specialized NusG paralog RfaH also reprograms virulence genes but regulates transcription elongation. To understand how elongation regulators might affect genes silenced by H-NS, we mapped H-NS, StpA (an H-NS paralog), RfaH, σ70, and RNA polymerase (RNAP) locations on DNA in the uropathogenic E. coli strain CFT073. Although H-NS-StpA filaments bind only 18% of the CFT073 genome, all loci at which RfaH binds RNAP are also bound by H-NS-StpA and are silenced when RfaH is absent. Thus, RfaH represents a distinct class of counter-silencer that acts on elongating RNAP to enable transcription through repressive nucleoprotein filaments. Our findings define a new mechanism of elongation counter-silencing and explain how RfaH functions as a virulence regulator.

Dataset Information

Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli.

Publications

Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli.

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