Author : Erik Dean Jessen
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (14 download)
Book Synopsis Local- and Genome-scale Study of the Interplay Between Escherichia Coli RNA Polymerase and Nucleoid-associated Proteins by : Erik Dean Jessen
Download or read book Local- and Genome-scale Study of the Interplay Between Escherichia Coli RNA Polymerase and Nucleoid-associated Proteins written by Erik Dean Jessen and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Bacterial transcription, once thought to be organized into discrete, largely non-overlapping units, has been revealed by deep cDNA sequencing to generate ubiquitous, overlapping, sense and antisense RNAs, many of which are noncoding. The function of the extensive antisense transcription of bacterial genes is controversial, and unclear in lieu of mechanistic dissection. We report here characterization of a model antisense transcription unit (bglAS) in the cryptic, H-NS-silenced b-glucoside utilization operon of E. coli K-12 (bglGFBH). bglAS was discovered because inhibition of Rho greatly increased its level and length. We created bglAS- alleles with little or no effect on bglF encoded in the sense strand by disabling the bglAS promoter with base substitutions. Although bglAS exists in a small H-NS-free island in the otherwise H-NS-coated bgl operon, the H-NS distribution is unchanged in bglAS- strains. However, when bglGFBH sense transcription was activated, bglAS decreased b-glucoside-induction of bgl gene expression via BglG-mediated antitermination of the bgl operon attenuators. Using a time series of ChIP-chip, we found that RNA polymerase progression through bglGFBH is hindered by bglAS transcription. In contrast, overexpression of bglAS RNA in trans had no effect on bgl operon induction, supporting bglAS function by transcriptional interference with bglGFBH expression. The bglAS promoter was upregulated by nitrite, consistent with bioinformatic detection of adjacent NarL/P binding sites and suggesting potential bglAS function as an environmental modulator. The proximity of bglAS to the surrounding H-NS filaments led us to investigate the interactions between transcription and H-NS. Consistent with the inability of transcription to affect H-NS binding patterns at the bgl operon, the genome-scale H-NS distribution exhibited minimal change when all transcription was inhibited with rifampicin. However, deletion of H-NS and StpA, an H-NS paralog, resulted in increased progression of RNAP along active, H-NS bound transcription units. Analyzing NET-seq data suggested that H-NS inhibits RNAP progression by promoting pausing of RNAP at non-canonical pause sequences. In contrast to H-NS, the nucleoid-associated protein HU was found to have a distribution pattern mirroring highly active transcription units. Upon further investigation, HU most closely correlated with the presence of R-loops, stable RNA:DNA hybrids external of RNAP. R-loop levels are independent of the presence of HU, suggesting HU is not involved in the resolution of R-loops. Instead, deletion of HU confers sensitivity to reactive oxygen species, and we propose a model by where HU binds to the areas around R-loops to protect DNA from mutagenesis. The combined studies presented here are a significant advancement in our understanding of the function of antisense transcripts and the interaction between transcription and nucleoid-associated proteins.
