Transcribing Activities in N4 Infected E. Coli

N4 感染大肠杆菌中的转录活动

• 批准号: 7742799
• 负责人: LUCIA B. B ROTHMAN-DENES
• 金额: $ 63.15万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7742799
• 关键词: Address; Alzheimer' s Disease; Amino Acids; Area; Bacteriophage N4; Bacteriophages; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biological Assay; C-terminal; Cell Death Signaling Process; Chloroplasts; Code; Collaborations; Complex; DNA; DNA Gyrase; DNA Sequence Rearrangement; DNA-Directed RNA Polymerase; Data; Degenerative polyarthritis; Disease; Dissection; Elongation Factor; Enzymes; Escherichia coli; Evolution; Family; Foundations; Gene Expression; Genes; Genetic Transcription; Genome; Grant; Heating; Infection; Injection of therapeutic agent; Laboratories; Lead; Length; Malignant Neoplasms; Mammalian Cell; Mitochondria; Mitochondrial RNA; Molecular; Molecular Conformation; Mutation; N-terminal; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Parkinson Disease; Pathway interactions; Peptides; Plasmids; Play; Polymerase; Polyproteins; Positioning Attribute; Protein Engineering; Proteins; RNA; RNA Polymerase III; Recruitment Activity; Refractory; Reporter; Reporting; Research; Resolution; Roentgen Rays; Role; SS DNA BP; Signal Pathway; Signal Transduction; Single-Stranded DNA; Site; Site-Directed Mutagenesis; Specificity; Structure; Structure-Activity Relationship; T7 RNA polymerase; Transcription Elongation; Transcription Initiation; Transcription Process; Transcriptional Activation; Virion; Work; Yeasts; base; improved; in vivo; interdisciplinary approach; member; multidisciplinary; polypeptide; promoter; protein crosslink; public health relevance; research study; scaffold; sensor; transcription factor

DESCRIPTION (provided by applicant): The proposed research represents our longstanding efforts to understand the structure, mechanisms of promoter recognition, polymerase and promoter activation, and transcription initiation and elongation of two phage N4-coded RNA polymerases (RNAP), vRNAP and N4 RNAPII. These RNAPs belong to the T7-like "single-subunit" RNAP family that includes mitochondrial RNAPs. In contrast to the archetypal T7 RNAP, the N4-encoded and mitochondrial RNAPs require protein factors throughout the transcription cycle. We recently reported the X-ray crystal structures of the N4 mini-vRNAP, the catalytic component of vRNAP, in the apo-form and hairpin-form promoter bound and transcription initiation complexes. This represents a major breakthrough and provides the foundation for addressing questions on the transcription mechanism of factor-dependent single-subunit RNAPs using multidisciplinary approaches as described below. 1. Functional and structural dissection of the vRNAP polypeptide: The vRNAP apo-form is in an inactive conformation, and undergoes a structural rearrangement that leads to activation upon promoter binding. We will identify the "sensor" in vRNAP and the "signal" in the hairpin-form promoter that lead to vRNAP activation using biochemical approaches. In addition, we will solve the structure of mini-vRNAP in the transition from apo-form to binary complex by improving preliminary crystals that diffract to 3.4 A resolution. Mini-vRNAP uses a transcription elongation factor, EcoSSB, to displace the RNA product from the DNA template. We will identify the EcoSSB site of interaction in the transcription elongation complex (TEC) using site-specific protein-protein crosslinking followed by site-directed mutagenesis. In contrast to T7 RNAP, mini-vRNAP is refractory to loading the DNA/RNA scaffold for TEC formation. Therefore, we will use structure-based protein engineering and the unique heat plasticity of vRNAP to prepare the mini-vRNAP TEC with DNA/RNA scaffold, and solve its structure with or without a EcoSSB peptide. 2. Elucidate the N4 RNAPII mechanism of promoter recognition: N4 RNAPII uses a transcription factor N4gp2, a single-stranded DNA binding protein, to recognize its promoters, which are similar in position and length to yeast mitochondrial RNAP promoters. Using an in vivo reporter assay, we will identify the N4 RNAPII determinants of promoter recognition by isolating N4 RNAPII derivatives that suppress promoter mutations. We will define the gp2 interaction platform on N4 RNAPII by site-specific protein-protein crosslinking followed by site-directed mutagenesis. We will also probe the role of gp2 in N4 RNAPII transcription elongation. To obtain a structural basis for factor-dependent single-subunit RNAP transcription, we will solve the crystal structures of the N4 RNAPII apo-form, promoter bound and TEC with or without gp2. We have recently identified and additional protein, N4gp1, required for N4 RNAPII transcription. We will determine its localization on the template in vivo, purify gp1 and analyze its effect on N4 RNAP transcription initiation. PUBLIC HEALTH RELEVANCE: The transcription process by bacteriophage N4-coded RNA polymerases has intriguing similarity to that found in mitochondria. Therefore, our multidisciplinary study of N4 RNA polymerases and transcription will provide a framework for understanding gene expression in mitochondria that generates over 90 % of the energy used by mammalian cells and also plays a key role in cell death signaling pathways and numerous disease states (Alzheimer's and Parkinson's diseases, osteoarthritis, type 2 diabetes mellitus, and cancer).

描述（由申请人提供）：拟议的研究代表了我们长期以来为理解两种噬菌体 N4 编码的 RNA 聚合酶 (RNAP)、vRNAP 和 N4 RNAPII 的结构、启动子识别机制、聚合酶和启动子激活以及转录起始和延伸所做的努力。这些 RNAP 属于 T7 样“单亚基”RNAP 家族，该家族包括线粒体 RNAP。与典型的 T7 RNAP 相比，N4 编码的 RNAP 和线粒体 RNAP 在整个转录周期中都需要蛋白质因子。我们最近报道了 N4 mini-vRNAP（vRNAP 的催化成分）在 apo 型和发夹型启动子结合和转录起始复合物中的 X 射线晶体结构。这代表了一项重大突破，并为使用下文所述的多学科方法解决因子依赖性单亚基 RNAP 转录机制的问题提供了基础。 1. vRNAP 多肽的功能和结构剖析：vRNAP apo 形式处于非活性构象，并经历结构重排，导致启动子结合后激活。我们将使用生化方法识别 vRNAP 中的“传感器”和发夹型启动子中导致 vRNAP 激活的“信号”。此外，我们将通过改进衍射分辨率达到3.4 A的初步晶体来解决mini-vRNAP从apo形式到二元复合物转变过程中的结构。 Mini-vRNAP 使用转录延伸因子 EcoSSB 来替换 DNA 模板中的 RNA 产物。我们将使用位点特异性蛋白质-蛋白质交联，然后进行定点诱变来识别转录延伸复合物 (TEC) 中的 EcoSSB 相互作用位点。与 T7 RNAP 相比，mini-vRNAP 难以加载用于 TEC 形成的 DNA/RNA 支架。因此，我们将利用基于结构的蛋白质工程和vRNAP独特的热可塑性来制备具有DNA/RNA支架的mini-vRNAP TEC，并在有或没有EcoSSB肽的情况下解析其结构。 2.阐明N4 RNAPII启动子识别机制：N4 RNAPII利用转录因子N4gp2（一种单链DNA结合蛋白）来识别其启动子，该启动子的位置和长度与酵母线粒体RNAP启动子相似。使用体内报告基因测定，我们将通过分离抑制启动子突变的 N4 RNAPII 衍生物来鉴定启动子识别的 N4 RNAPII 决定因素。我们将通过位点特异性蛋白质-蛋白质交联，然后进行定点诱变来定义 N4 RNAPII 上的 gp2 相互作用平台。我们还将探讨 gp2 在 N4 RNAPII 转录延伸中的作用。为了获得因子依赖性单亚基 RNAP 转录的结构基础，我们将解析 N4 RNAPII apo 形式、启动子结合以及有或没有 gp2 的 TEC 的晶体结构。我们最近发现了 N4 RNAPII 转录所需的额外蛋白质 N4gp1。我们将确定其在体内模板上的定位，纯化 gp1 并分析其对 N4 RNAP 转录起始的影响。公共健康相关性：噬菌体 N4 编码的 RNA 聚合酶的转录过程与线粒体中发现的转录过程具有有趣的相似性。因此，我们对 N4 RNA 聚合酶和转录的多学科研究将为理解线粒体中的基因表达提供一个框架，线粒体产生哺乳动物细胞所用能量的 90% 以上，并且在细胞死亡信号通路和多种疾病状态（阿尔茨海默氏症）中发挥着关键作用。和帕金森病、骨关节炎、2 型糖尿病和癌症）。