Cellular factors maintaining and reversing silencing of bacterial chromatin

All living cells possess mechanisms that partition the chromosomal DNA into actively transcribed, accessible domains, and transcriptionally silent domains densely packed by DNA-bound proteins. In Escherichia coli, nucleoid-associated proteins (NAPs) assemble into nucleoprotein filaments that cover several kilobase-long regions of DNA, silencing expression of horizontally acquired xenogenes, virulence factors or enzymes needed for utilization of exotic nutrients. While silencing of promoters, frequently by exclusion of RNA polymerase, is well studied, silencing during RNA chain elongation has only recently come to light. E. coli H-NS and its homolog StpA are NAPs that directly bind to AT-rich DNA and inhibit transcription initiation and elongation. The available data show that the transcription elongation complex is involved in both maintenance and relief of NAP-mediated silencing. Recent data implicate the ω subunit of RNA polymerase in regulation of global DNA topology and transcription of xenogeneic regions. A universally conserved elongation factor, NusG, and termination factor Rho, which are associated with RNA polymerase genome-wide and stop synthesis of RNAs that are not actively translated, cooperate with H-NS to silence xenogenes and other inactive genes. Conversely, we showed that a specialized NusG paralog, RfaH, which is required for expression of xenogeneic operons, excludes NusG and Rho from the transcribing RNA polymerase and counteracts NAP-mediated silencing. We propose to elucidate molecular mechanisms which control the accessibility of bacterial chromatin, focusing on poorly understood regulation during transcription elongation. In Aim 1, we will investigate effects of the ubiquitous ω subunit on RNA polymerase structure and activity. We will identify cellular factors that interact with ω using genetics and proteomics, characterize ω-induced changes in transcription complexes, and investigate ω effects on in vitro RNA synthesis. In Aim 2, we will study regulation of Rho- dependent termination. We posit that hyperactive Rho may be harmful during slow growth or translational stress and will investigate mechanisms by which Rho activity may be globally downregulated, e.g., by changing Rho conformation, promoting the formation of inactive Rho filaments, or blocking Rho binding to RNA. In Aim 3, we will identify new factors that contribute to maintenance of E. coli heterochromatin; determine contributions of different NAPs, Rho, and ω to silencing; and elucidate the molecular mechanism by which RfaH counter-silences NAPs in vitro.