CONTROL MECHANISMS IN TEMPERATE BACTERIOPHAGE LAMBDA

温带噬菌体 Lambda 的控制机制

• 批准号: 6107968
• 负责人: Robert A Weisberg
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6107968
• 关键词: DNA directed RNA polymerase; bacteriophage lambda; genetic recombination; genetic transcription; integrase; microorganism culture; nucleic acid sequence; recombinase; suppressor mutations; transcription factor; virus genetics; virus infection mechanism

Anti-termination increases the transcription of genes that are located downstream of terminators. Transcripts encoded by the cis-acting antitermination sites (put) of lambdoid phage HK022 promote readthrough of multiple downstream transcription terminators. Efficient readthrough depends on the structure of the put transcript and of a domain located in the largest subunit of RNA polymerase, suggesting that an interaction between put RNA and this domain is a step in anti-termination. To study put structure and function, we characterized a large number of mutants and probed synthetic transcripts with structure-specific nucleases. The results argue that the secondary structure of active put RNA consists of two hairpin stems that are separated by a single unpaired base. Internal loops and bulges in one of the stems are important for activity. Some variation in the lengths of the stems, the sizes and sequences of the terminal loops, and the locations of the internal loops and bulges is allowed. We propose that put acts by preventing dissociation of the ter nary transcription complex at terminator sites, because a terminator that arrests polymerase but does not cause dissociation is resistant to put. The integrase proteins of phages lambda and HK022 are closely related site- specific recombinases that recognize different nucleotide sequences in the core regions of their substrates, the attachment sites of the two phages. In lambda attachment sites, -2C, -1T, and +4G of the B' extended core binding site and -1T of C are major anti-HK022 determinants. In HK022 attachment sites, +1A and +3C of B' and C are major anti- determinants. These determinants are best viewed as negative because when they were replaced with the corresponding bases from the other attachment site, specificity was relaxed. Residues at certain positions within the two integrases were identified as specificity determinants. We have now shown that the phenotypes of amino acid substitutions at these positions can be suppressed in an allele-specific manner by nucleotide substitutions in the attachment sites, suggesting a direct and specific amino acid-nucleotide interaction.

抗终止增加了所在基因的转录 终结者的下游。 由顺式作用编码的笔录 Lambdoid Phage HK022的抗释放位点（PUT）促进读取 多个下游转录终止剂。 有效的读取 取决于PUT转录本的结构和位于 RNA聚合酶的最大亚基，表明相互作用 在PUT RNA和该域之间是抗终止的一步。 学习 放置结构和功能，我们表征了大量突变体 并用结构特异性核酸酶进行探测的合成转录本。 这 结果认为，主动put RNA的二级结构由 两个由单个未配对的底座隔开的发夹茎。 内部的 其中一个茎中的环和凸起对于活动很重要。 一些 茎长度，大小和序列的变化 终端环，内部环和凸起的位置为 允许。 我们建议通过防止Ter解离来提出行为 终结器站点的Nary转录复合物，因为终结者 逮捕聚合酶，但不会引起解离是抗药性的。 噬菌体lambda和hk022的整合酶蛋白密切相关 识别不同核苷酸序列的位点特异性重组酶 在其底物的核心区域，两者的附件位点 噬菌体。 在lambda附件站点中，-2C，-1T和 +4G的B'扩展 C的核心结合位点和-1T是主要的抗HK022决定因素。 在 B'和C的HK022附件 +1a和 +3c是主要抗 决定因素。 这些决定因素最好被视为负面，因为 当它们被另一个的相应基础替换时 附着位置，特异性放松。 残留在某些位置 在两个集成酶中，被确定为特异性决定因素。 现在，我们已经表明氨基酸取代的表型 这些位置可以通过特定于等位基因的方式来抑制 附着位点的核苷酸取代，表明直接和直接 特定的氨基酸核苷酸相互作用。