The bacterial chromosome is condensed over 1000- fold through its compaction and organization by nucleoid proteins [3,4]. One of the most studied members of the nucleoid protein family is heat-stable nucleoid structuring protein (H-NS). H-NS binds to A/T rich sequences and can nucleate vast stretches of DNA through oligimerization . Due to promoters having high A/T content as well, H-NS has been shown to bind to promoters and repress the transcription of <5% of E. coli genes . Recent ChIP- chip studies of H-NS have shown that this protein can bind to highly transcribed genes in their coding regions, suggesting that elongating RNA polymerase (RNAP) interacts with H-NS during transcription . To date, there is little known about the effects that H-NS has on an elongating RNAP when they encounter each other. However, in eukaryotes the effects of an elongating RNA pol II into nucleosomes are well studied, and nucleosomes have been shown to cause RNA pol II backtracking [19-25].
I aim to uncover what happens when an elongating RNAP transcribes into H-NS bound DNA. Recent work from our lab found that the cryptic bgl operon has an antisense transcript that originates from the bglF gene, and transcription that initiates from the antisense promoter presumably runs directly into an H-NS patch . In my first aim I plan to identify and mutate the antisense promoter. Mutant cell lines will be tested for the ability activates the bgl operon through 2-glucosidase assays to see if this antisense promoter is important in regulating the activation of the bgl operon. In the second aim, I plan recapitulate an elongating RNAP transcribing into H-NS patches in vitro, using an in vitro reconstitution assay. This type of assay has been used to test the effects of an elongating RNA pol II transcribing into nucleosomes in vitro [21, 25]. Once this system has been optimized, I can start to understand the mechanism of the transcription barrier that H-NS has on RNAP. Additionally, we can add elongation factors to the assay to understand if they help or inhibit RNAP transcribe through H-NS bound DNA. To validate the data gained through the in vitro reconstitution assay, in the third aim I plan to use ChIP-chip to understand the prevalence of RNAP encountering nucleoid proteins at a genome level. This work will start to uncover the effects H-NS has on an elongating RNAP, and its frequency and significance in regulating gene expression in E. coli.
The nucleoid protein H-NS regulates the expression of numerous virulence genes along with horizontally acquired genes in enterobacteria, both of which cause significant threats to human health [36, 47, 50-54]. To date there is little known about how RNAP interacts with H-NS bound DNA. Understanding the effect that H-NS has on an elongating RNAP could provide a new therapeutic avenue to combat pathogenic enterobacteria or the spread of antibiotic resistance genes .
|Kotlajich, Matthew V; Hron, Daniel R; Boudreau, Beth A et al. (2015) Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria. Elife 4:|