RNAP-binding proteins encoded by Thermus phages

栖热菌噬菌体编码的 RNAP 结合蛋白

• 批准号: 7294085
• 负责人: Leonid Minakhin
• 金额: $ 19.31万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294085
• 关键词: Affect; Affinity; Anti-Bacterial Agents; Antibiotics; Bacteriophages; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Collaborations; Complex; DNA-Directed RNA Polymerase; Depth; Drug Delivery Systems; Enzymes; Eubacterium; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; In Vitro; Infection; Laboratories; Life; Mediating; Molecular; Molecular Mechanisms of Action; Organism; Pharmaceutical Preparations; Process; Protein Analysis; Protein Binding; Proteins; RNA Polymerase Inhibitor; Recombinants; Regulation; Rifampin; Site; Staging; System; Thermus; Transcriptional Regulation; Tuberculosis; Viral; Virus; Work; antibiotic design; cellular targeting; drug development; genetic regulatory protein; novel; protein protein interaction; thermophilic bacteria; thermophilic organism; transcription factor

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.

描述（由申请人提供）：RNA聚合酶（RNAP）是转录的中心酶，也是调节的主要靶点。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（Bacteriophage，简称BPHs）进化出有效的调控机制来转换宿主的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多样的生命形式，噬菌体编码的转录因子的范围实际上是取之不尽的。这些噬菌体编码的蛋白质的研究提供并继续提供一般生物学意义的遗传调控的范例。研究嗜热菌（其蛋白质形成上级晶体的生物体）的噬菌体感染具有额外的优势，因为它们使得有可能获得关于噬菌体编码的调节剂与其细胞靶点RNAP复合的结构信息。我们的目标是研究噬菌体依赖的转录调控嗜热栖热真细菌的RNAP已结晶。尽管噬菌体基因组学最近取得了进展，但只有少数嗜热噬菌体被完全测序。近年来，对3株感染T.嗜热菌（Tth）。Tth感染的过程中，每一个这些病毒将被研究，以实现以下具体目标。1.与Tth RNAP相互作用的噬菌体编码的蛋白质将通过在高效免疫亲和纯化期间与亲和标记的宿主RNAP共纯化的蛋白质的质谱分析来鉴定。2.已鉴定的噬菌体蛋白将在我们开发的完全重组Tth RNAP体外转录系统中进行功能表征;噬菌体调节剂及其在宿主RNAP上的结合位点的分子作用机制将被确定。为了更深入地理解转录调控的机制，我们将对噬菌体调节因子和宿主RNAP之间的复合物进行结构分析。结构工作将与两个领先的晶体学小组合作进行。作为拟议的合作研究的结果，新的噬菌体编码的RNAP结合转录蛋白将被确定和功能和结构的特点。研究结果将揭示新的转录调控机制，并揭示RNAP位点，这些位点可用作开发药物的潜在靶点，这些药物通过影响细菌RNAP（一种经验证的药物靶点）发挥作用。噬菌体进化出一系列非常多样的调节蛋白，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，并且是经验证的抗菌药物靶标：利福平，RNAP抑制剂，目前是抗结核病的一线药物。因此，结合细菌RNAP并抑制基因表达的噬菌体蛋白的鉴定和表征将具有鉴定新抗生素蛋白和合理有效的抗生素设计的巨大潜力。