RNAP-binding proteins encoded by Thermus phages

描述(申请人提供)：RNA聚合酶(RNAP)是转录的中心酶，也是调节的主要目标。转录周期的每个阶段都可以通过RNAP与各种转录因子的相互作用来调节。噬菌体进化出有效的调节机制来转换宿主转录机制以满足噬菌体的需要。由于噬菌体是生物圈中最丰富和最多样化的生命形式，因此噬菌体编码的转录因子的范围几乎是取之不尽的。对这类噬菌体编码蛋白的研究提供并继续提供具有普遍生物学意义的遗传调控范例。对感染嗜热细菌的噬菌体的研究有一个额外的优势，因为它们使获得与其细胞靶标RNAP复合的噬菌体编码调节子的结构信息成为可能。我们的目标是研究RNAPs已经结晶的嗜热嗜热菌真细菌中依赖于噬菌体的转录调控。尽管最近在噬菌体基因组学方面取得了进展，但只有几个嗜热噬菌体被完全测序。最近，我们实验室对感染嗜热菌的3株噬菌体的基因组进行了测定。将对这些噬菌体感染TTH的过程进行研究，以实现以下具体目标。1.与TTH RNAP相互作用的噬菌体编码蛋白将通过与亲和标记的宿主RNAP在高效免疫亲和纯化过程中共纯化的蛋白质的质谱分析来鉴定。2.利用我们建立的全重组TTH RNAP体外转录系统对已鉴定的噬菌体蛋白进行功能鉴定，确定噬菌体调节剂及其与宿主RNAP结合的分子机制。为了更深入地了解转录调控机制，我们将对噬菌体调控因子与宿主RNAP之间的复合体进行结构分析。结构工作将与两个主要的结晶学小组合作进行。作为拟议的合作研究的结果，新的噬菌体编码的RNAP结合转录蛋白将被鉴定并从功能和结构上进行表征。这一结果将揭示新的转录调控机制，并将揭示RNAP位点可用作开发通过影响细菌RNAP的药物的潜在靶点，RNAP是一个有效的药物靶点。噬菌体进化出一系列非常多样化的调节蛋白，这些蛋白结合并抑制细菌RNA聚合酶(RNAP)。细菌RNAP是基因表达的中心酶，也是有效的抗菌药物靶点：RNAP抑制剂利福平目前是抗结核的一线药物。因此，鉴定和鉴定与细菌RNAP结合并抑制基因表达的噬菌体蛋白，将对鉴定新的抗生素蛋白和合理有效的抗生素设计具有巨大的潜力。

DESCRIPTION (provided by applicant): RNA polymerase (RNAP) is the central enzyme of transcription and a major target of regulation. Each stage of the transcription cycle can be regulated through interactions of RNAP with various transcription factors. Bacteriophages (phages) evolved efficient regulatory mechanisms to convert host transcription machinery to serve the needs of the phage. Since phages are the most abundant and diverse life form in the Biosphere, the gamut of phage-encoded transcription factors is practically inexhaustible. Studies of such phage-encoded proteins provided and continue to provide paradigms of genetic regulation of general biological significance. Studies of phages infecting thermophilic bacteria, organisms whose proteins form superior crystals, have an added advantage, for they make it possible to obtain structural information about phage-encoded regulators in complex with their cellular target, RNAP. Our goal is to study bacteriophage-dependent regulation of transcription in thermophilic Thermus eubacteria whose RNAPs have been crystallized. Despite recent advances in phage genomics, only a few thermophilic phages have been completely sequenced. Recently, the genomes of 3 phages infecting T. thermophilus (Tth) were determined in our laboratory. The process of Tth infection by each of these phages will be studied to achieve the following specific aims. 1. Phage-encoded proteins that interact with Tth RNAP will be identified by mass- spectrometric analysis of proteins that co-purify with affinity-tagged host RNAP during a highly-efficient immunoaffinity purification. 2. Identified phage proteins will be functionally characterized in a fully recombinant Tth RNAP in vitro transcription system that we developed; the molecular mechanisms of action of the of phage regulators and their binding sites on host RNAP will be determined. To deeper understand the mechanism of transcription regulation we will perform structural analysis of complexes between phage regulators and host RNAP. The structural work will be carried out as a collaboration with two leading crystallographic groups. As a result of proposed collaborative studies, novel phage-encoded RNAP-binding transcription proteins will be identified and characterized functionally and structurally. The results will uncover novel transcription regulation mechanisms and will reveal RNAP sites that can be used as potential targets for development of drugs that act by affecting bacterial RNAP, a validated drug target. Bacteriophages evolved a remarkably diverse array of regulatory proteins that bind to and inhibit bacterial RNA polymerase (RNAP). Bacterial RNAP is a central enzyme in gene expression and is a validated antibacterial drug target: Rifampicin, an RNAP inhibitor, currently is a frontline drug against tuberculosis. Thus, identification and characterization of phage proteins that bind bacterial RNAP and inhibit gene expression will have a tremendous potential for identifying new antibiotic proteins and for rational and effective antibiotic design.