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Nascent elongating transcript sequencing (NET-seq) for Escherichia coli and Bacillus subtilis reveals a consensus pause sequence enriched at translation start sites.

ABSTRACT: Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing we identify a 16 nt consensus pause sequence in E. coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP/nucleic-acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and B. subtilis. Our results thus implicate a conserved mechanism unifying known and newly identified pause events.

ORGANISM(S): Escherichia coli Bacillus subtilis

PROVIDER: GSE56720 | GEO | 2014/05/05

SECONDARY ACCESSION(S): PRJNA244362

REPOSITORIES: GEO

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Project description:Recent genome-scale ChIP-chip studies of transcription factors have shown that a low percentage of experimentally determined binding sites contain the consensus motif for the immunoprecipitated factor. In most cases, differences between in vivo target sites that contain or lack a consensus motif have not been explored. We have previously shown that most sites to which E2F family members are bound in vivo do not contain E2F consensus motifs. The main purpose of this study was to develop an understanding of how E2F binding specificity is achieved in vivo. In particular, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using promoter and ENCODE arrays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are a) the site must be in a core promoter and b) the promoter region must be utilized as a promoter by the transcriptional machinery in that particular cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including a) indirect recruitment, b) looping to the core promoter mediated by an E2F bound to a distal consensus motif, and c) assisted binding of E2F to a site that weakly resembles an E2F consensus motif within the core promoter. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated promoter fragments. Our findings suggest that in vivo a) the presence of a consensus motif is not sufficient to recruit E2Fs, b) E2Fs can bind to isolated regions that lack a consensus motif, and c) binding can require regions other than the best match to the E2F PWM in the core promoter. Keywords: E2F, ChIP-chip, transcription factor binding, consensus motifs 37 ChIP-chip arrays (of these, 14 array sets are biological duplicates). 22 samples are included in this series, the rest can be found in supplementary info to the following papers: Xu 2007, Jin 2006, Komashko 2008

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Nascent elongating transcript sequencing (NET-seq) for Escherichia coli and Bacillus subtilis reveals a consensus pause sequence enriched at translation start sites.

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