Reactivity epitopes of ribonuclease III substrates

核糖核酸酶 III 底物的反应性表位

• 批准号: 7322254
• 负责人: Allen Whitall Nicholson
• 金额: $ 27.02万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7322254
• 关键词: 2-Aminopurine; Affinity; Antibiotics; Bacteriophage lambda; Bacteriophages; Base Pairing; Binding; Binding Sites; Circular Dichroism; Cleaved cell; Communicable Diseases; Complex; Data; Defense Mechanisms; Dependence; Disease; Double-Stranded RNA; E coli ribonuclease III; Elements; Endoribonucleases; Enzymes; Epitopes; Escherichia coli; Eukaryotic Cell; Event; Family; Fluorescence; Gene Expression; Gene Silencing; Genome; Goals; Half-Life; In Vitro; Ions; Maintenance; Malignant Neoplasms; Maps; Messenger RNA; MicroRNAs; Mitochondria; Mutation; Orthologous Gene; Pancreatic ribonuclease; Plasmids; Process; Production; Proteins; Protocols documentation; RNA; RNA Binding; RNA Interference; RNA Sequences; RNA-Protein Interaction; Range; Reaction; Regulator Genes; Reporter; Reporter Genes; Research Personnel; Resistance; Ribonuclease III; Ribonucleases; Role; Side; Site; Site-Directed Mutagenesis; Sodium Chloride; Specificity; Structure; Substrate Specificity; System; Thermotoga maritima; Translations; Trypanosoma; Virulence Factors; base; defined contribution; divalent metal; endoribonuclease; functional group; human DICER1 protein; in vivo; insight; member; nucleotide analog; polypeptide; programs; research study; translation assay

DESCRIPTION (provided by applicant): The enzymatic cleavage of double-stranded RNA is an essential step in the maturation and degradation of diverse eukaryotic and prokaryotic RNAs, and is a key event in RNA interference (RNAi) and related gene silencing and genome maintenance mechanisms. dsRNA cleavage is catalyzed by members of the ribonuclease III (RNase III) family of endoribonucleases, which are highly conserved in bacterial and eukaryotic cells. Eukaryotic RNase III orthologs include Dicer and Drosha, which carry out the maturation of microRNAs and other gene-regulatory RNAs. RNase III also is a component of RNA editosomes in trypanosome mitochondria. dsRNA processing by bacterial ribonucleases III regulates cellular and phage gene expression, plasmid replication, antibiotic production, and virulence factor expression. Bacterial RNase substrates are cleaved in a highly site-specific manner, which is required for proper half-life or optimal function of the RNAs. There also is evidence that bacterial RNase III can regulate gene expression by binding RNA in a non-catalytic mode. The long-term objective of this project is to characterize the RNA sequence and structural elements and RNA-protein interactions that confer specificity in RNase III substrate recognition and cleavage. Specific Aim 1 will employ in vitro selection, nucleotide analog interference mapping and site-directed mutagenesis to identify substrate functional groups essential for recognition. Specific Aim 2 will characterize protein functional group contributions to substrate specificity and binding energy. Site-directed mutagenesis and polypeptide segment exchange experiments will identify RNase III domains involved in specificity of binding. Specific Aim 3 will define the sequence and structural features of catalytic antideterminants in two specific RNAs. In vitro selection, site-directed mutagenesis, and RNA structure-probing will be used to determine the critical structural and functional features of an in vitro-selected, binding-competent RNA, and the lambda phage cIII mRNA 5'-leader. The mechanism of RNase III activation will be analyzed using an in vitro translation system. Along with Escherichia coli RNase III, this project will employ Aquifex aeolicus RNase III and Thermotoga maritima RNase III, for which structural data is available. These analyses will serve to define how specificity is achieved in dsRNA processing by RNase III, and ultimately determine the role of dsRNA processing in the cellular defense mechanisms, including RNAi and in specific disease states, including cancer and infectious diseases.

描述（由申请人提供）：双链RNA的酶促切割是多种真核和原核RNA成熟和降解的重要步骤，是RNA干扰（RNAi）和相关基因沉默和基因组维持机制的关键事件。双链RNA切割由核糖核酸酶III（RNase III）家族的内切核糖核酸酶成员催化，该家族在细菌和真核细胞中高度保守。真核RNase III直系同源物包括Dicer和Drosha，它们进行microRNA和其他基因调控RNA的成熟。RNase III也是锥虫线粒体中RNA编辑体的组分。由细菌核糖核酸酶III进行的dsRNA加工调节细胞和噬菌体基因表达、质粒复制、抗生素产生和毒力因子表达。细菌RNA酶底物以高度位点特异性的方式切割，这是RNA的适当半衰期或最佳功能所需的。还有证据表明，细菌RNase III可以通过以非催化模式结合RNA来调节基因表达。该项目的长期目标是表征RNA序列和结构元件以及赋予RNase III底物识别和切割特异性的RNA-蛋白质相互作用。具体目标1将采用体外选择，核苷酸类似物干扰作图和定点诱变，以确定识别所必需的底物官能团。特异性目标2将表征蛋白质官能团对底物特异性和结合能的贡献。定点诱变和多肽片段交换实验将鉴定参与结合特异性的RNase III结构域。特定目标3将定义两种特定RNA中催化反决定簇的序列和结构特征。将使用体外选择、定点诱变和RNA结构探测来确定体外选择的具有结合能力的RNA和λ噬菌体cIII mRNA 5 '-前导序列的关键结构和功能特征。将使用体外翻译系统分析RNase III激活的机制。沿着大肠杆菌RNase III，该项目将使用风产液菌RNase III和海栖热袍菌RNase III，其结构数据可用。这些分析将用于定义RNA酶III在dsRNA加工中如何实现特异性，并最终确定dsRNA加工在细胞防御机制中的作用，包括RNAi和特定疾病状态，包括癌症和传染病。