Cellular factors maintaining and reversing silencing of bacterial chromatin

所有活着的细胞都有将染色体DNA分割成活跃转录的机制， 可访问的结构域，以及被DNA结合蛋白密集包装的转录沉默结构域。在……里面 大肠埃希氏菌，类核相关蛋白(NAP)组装成覆盖在 DNA的几个千碱基长区域，水平获得性异种基因的沉默表达，毒力 利用外来营养所需的因子或酶。虽然让发起人保持沉默，通常是通过 排除RNA聚合酶，是很好的研究，沉默在RNA链延长只是最近 来揭露真相吧。大肠杆菌H-NS及其同系物StpA是直接与富含AT的DNA结合并抑制的NAP 转录起始和延伸。现有数据表明，转录延伸复合体 参与维持和缓解NAP介导的沉默。最近的数据表明ω RNA聚合酶亚基在全球DNA拓扑调控和异种区转录中的作用。 一种普遍保守的伸长因子NusG和终止因子Rho，它们与 RNA聚合酶全基因组并停止合成非主动翻译的RNA，合作 用H-NS来沉默异源基因和其他非活性基因。相反，我们展示了一个专门的 NusG paralog，RfaH是表达异源操纵子所必需的，不包括NusG和Rho 从转录的RNA聚合酶中分离出来，并抵消NAP介导的沉默。我们建议 阐明控制细菌染色质可及性的分子机制 了解转录延伸过程中的调节。在目标1中，我们将调查 聚合酶结构和活性中普遍存在的ω亚单位。我们将确定细胞因子， 使用遗传学和蛋白质组学与ω相互作用，表征ω诱导的转录变化 并研究ω对体外核糖核酸合成的影响。在目标2中，我们将研究Rho-的调节- 从属终止。我们推测，过度活跃的Rho在生长缓慢或转化过程中可能是有害的 应激，并将调查Rho活动可能在全球范围内下调的机制，例如 改变Rho构象，促进非活性Rho细丝的形成，或阻止Rho结合 致RNA。在目标3中，我们将确定有助于维持大肠杆菌异染色质的新因素； 确定不同的NAP、Rho和ω对沉默的贡献；并阐明分子 RfaH体外抑制NAPs的机制。

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.