Transcribing Activities in N4 Infected E. Coli

N4 感染大肠杆菌中的转录活动

• 批准号: 8291346
• 负责人: LUCIA B. B ROTHMAN-DENES
• 金额: $ 57.93万
• 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/8291346
• 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; research study; scaffold; sensor; transcription factor

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 ¿ 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.

拟议的研究代表了我们为了解结构和机制的长期努力 启动子识别，聚合酶和启动子激活以及两个的转录启动和伸长 噬菌体N4编码的RNA聚合酶（RNAP），VRNAP和N4 RNAPII。这些RNAP属于T7 包括线粒体rnap的“单单基因” rnap家族。与原型T7 RNAP相反， N4编码和线粒体RNAP在整个转录周期中需要蛋白质因子。我们最近 报道了Apo形式中N4 Mini-VRNAP的X射线晶体结构，VRNAP的催化成分 和发夹形式的启动子结合和转录倡议复合物。这代表了一个重大突破 并为解决因子依赖性转录机理的问题提供了基础 如下所述，使用多学科方法的单单基因RNAP。 1。VRNAP多肽的功能和结构解剖：VRNAP APO形式在不活跃中 构象，并经历结构重排，从而导致启动子结合的激活。我们 将识别VRNAP中的“传感器”和导致VRNAP激活的发夹形式启动子中的“信号” 使用生化方法。此外，我们将在从apo-的过渡中解决微型vrnap的结构 通过改善衍射为3.4»分辨率的初步晶体来形成二进制复合物。迷你VRNAP使用 转录伸长因子ECOSSB，以从DNA模板中置换RNA产物。我们将确定 使用位点特异性蛋白质蛋白的转录伸长复合物（TEC）中的ECOSSB相互作用位点 交联，然后进行位置定向诱变。与T7 RNAP相比 为TEC形成加载DNA/RNA支架。因此，我们将使用基于结构的蛋白质工程 VRNAP的独特热可塑性，用DNA/RNA支架准备小型VRNAP TEC，并解决其 带有或没有Ecossb辣椒的结构。 2。阐明启动子识别的N4 RNAPII机制：N4 RNAPII使用转录因子 N4GP2，一种单链DNA结合蛋白，以识别其启动子，其位置相似，并且 酵母线粒体RNAP启动子的长度。使用体内记者测定法，我们将识别N4 RNAPII 通过分离抑制启动子突变的N4 RNAPII衍生物，启动子识别的决定因素。我们 将通过位点特异性蛋白质 - 蛋白交联，然后定义N4 RNAPII上的GP2相互作用平台 定向诱变。我们还将探测GP2在N4 RNAPII转录伸长率中的作用。获得 依赖因子依赖的单人含量RNAP转录的结构基础，我们将解决的晶体结构 N4 RNAPII APO形式，启动子绑定和TEC，带有或不带有GP2。我们最近确定了 N4 RNAPII转录所需的其他蛋白质N4GP1。我们将确定其本地化 模板在体内，净化GP1并分析其对N4 RNAP转录启动的影响。