TRANSFER RNAS AND THEIR GENES IN THE ARCHAEBACTERIA

古细菌中的 RNA 转移及其基因

• 批准号: 3289611
• 负责人: Ramesh Gupta
• 金额: $ 9.14万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289611
• 关键词: DNA directed RNA polymerase; Halobacteriaceae; Methanobacteriaceae; bacterial genetics; gene expression; genetic manipulation; genetic transcription; microorganism culture; nucleic acid sequence; nucleic acid structure; sulfur metabolizing bacteria; thermophilic organism; transfer RNA

The archaebacteria are distinct from the eubacteria and the eukaryotes. Though the basic life processes are similar in the three kingdoms, in details of molecular features and various functional mechanisms, the archaebacteria are either unique or resemble one or the other of the remaining two kingdoms. There are two main divisions of archaebacteria: (i) methanogen-extreme halophiles, and (ii) sulfur-utilizing thermophiles. The latter division more closely resembles eukaryotes than does the former. Therefore, the study of archaebacterial systems, besides having a comparative phylogenetic importance, could also be considered as a study of a eukaryote-like system at a simpler (prokaryotic) level, which might have evolved to a more complex (eukaryotic) level. The long term goals of this study are to understand the structure and function of transfer RNAs and the organization and expression of tRNA genes in archaebacteria. The specific aims of this proposal are the isolation and sequencing of Sulfolobus sp. tRNAs, characterization of modified nucleosides in archaebacterial tRNAs, determination of the sequences of Halobacterium volcanii tRNA genes and their flanking regions, determination of RNA polymerase binding sites in archaebacterial genes to identify promotor regions, and development of an in vitro RNA processing system for archaebacterial gene transcripts. The tRNAs will be sequenced by a combination of two-dimensional paper electrophoresis of RNAse digests of uniformly 32P-labeled tRNAs and gel sequencing methods using end-labeled tRNAs. Modified nucleosides will be identified by various electrophoretic, chromatographic, mass spectrometric and NMR techniques. The tRNA genes will be cloned in plasmid vectors and sequenced by dideoxy termination and chemical methods. RNA polymerase binding sites in promotor-containing DNA will be determined by the DNAse I "foot printing" technique. Pilot studies will be done to identify an RNA processing activity in crude cell extracts using either natural precursors or in vitro SP6 or T7 polymerase-transcribed RNAs from archaebacterial tRNA genes. Then attempts will be made to define and characterize the components involved in this processing system.

古细菌不同于真细菌和真核生物。 虽然三国的基本生命过程是相似的， 详细的分子特征和各种功能机制， 古细菌要么是独特的，要么类似于一个或另一个 剩下两个王国。 古细菌主要分为两大类： (i)产甲烷极端嗜盐菌，和（ii）硫利用嗜热菌。 后一个分裂比前一个分裂更接近真核生物。 前者 因此，古细菌系统的研究，除了有一个 比较系统发育的重要性，也可以被认为是一个研究 在一个更简单的（原核）水平上的类似真核生物的系统， 进化到更复杂的（真核）水平。 本研究的长期目标是了解结构， 转运RNA的功能和tRNA基因的组织和表达 在古细菌中。 这项提案的具体目的是隔离 Sulfolobus sp. tRNA的测序，修饰的 古细菌trna中的核苷序列测定 火山盐杆菌tRNA基因及其侧翼区的测定 的RNA聚合酶结合位点， 启动子区，并开发了一种体外RNA加工系统， 古细菌基因转录物。 tRNA将通过二维纸结合测序 均匀32P标记的tRNA的RNA酶电泳和凝胶电泳 使用末端标记的tRNA的测序方法。 修饰的核苷将是 通过各种电泳、色谱、质谱 和NMR技术。 tRNA基因将被克隆到质粒载体中， 通过双脱氧终止和化学方法测序。 RNA聚合酶 含有启动子的DNA中的结合位点将由DNA酶I “脚印”技术。 将进行初步研究，以确定一种RNA 在粗细胞提取物中使用天然前体的加工活性 或体外SP6或T7聚合酶转录的RNA 基因. 然后，将尝试定义和表征 这一过程中涉及到的各个环节。