GLOBAL RNA STRUCTURE AND GENE REGULATION

全球RNA结构和基因调控

• 批准号: 2186217
• 负责人: ANN B JACOBSON
• 金额: $ 18.92万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186217
• 关键词: coliphages; computer simulation; conformation; electron microscopy; gene expression; genetic regulatory element; genetic translation; genome; model design /development; molecular rearrangement; mutant; nucleic acid metabolism; nucleic acid sequence; physical model; protein biosynthesis; site directed mutagenesis; tissue /cell culture; virus RNA; virus genetics; virus protein

In this proposal, two hypotheses concerning the regulation of RNA coliphage gene expression by long range RNA-RNA interactions will be examined: 1) Stable long range interactions at the 5' end of Q beta RNA suppress transnational initiation of the viral maturation protein. (2) Two alternate conformations (CH and ACH) of a large structural feature, the central hairpin, serve as a structural switch for the regulations of transnational initiation of viral coat protein. The long range RNA secondary structures, located within coding regions of genomic RNA, have been identified in structural studies on the conformation of genomic and mutant RNAs of coliphage Qbeta by the principal investigator. Their location within the viral genetic map suggest their function in the regulation of transnational initiation. The structural studies have shown that genomic RNA from coliphage Qbeta (4217 nucleotides) can be divided into a series of large structural domains that are well determined, as well as several regions where predicted structures are unusually plastic. The well determined structural domains range in size from 500 to 700 nucleotides and include the initiation region for the translation of viral maturation protein at he 5' end of Qbeta RNA. Variable regions include the initiation region for the translation for the viral coat and viral replicase translation. These sites are located in the lower half of a central hairpin (1500 nucleotides) where the structural rearrangements between the CH and ACH conformations are observed. In the CH conformation, the coat initiation region is free and large amounts of viral coat protein could be synthesized. In the ACH conformation, the coat initiation region is buried in extensive secondary structure. Coat synthesis (and all other viral protein synthesis) would be switched off. The switch may be mediated by viral and/or cellular proteins, possibly by viral replicase. A large structural switch of this type is novel, and has not been proposed previously in any experimental system. The proposed analyses will proceed in several steps. Additional structural studies with native and mutant RNAs are needed to develop reliable structural models at the nucleotide level for the long range interactions both at the 5' end of the RNA and in the region of the CH- ACH switch. On the basis of these studies, mutant RNAs will be created in which the proposed long range interactions will have been disrupted. The effect of these structural changes will be studied both in vitro and in vivo. Several unusual tools will be used in these analysis of potential structural changes in full size genomes by energy dot plot, and a genetic system in which cis-acting structural defects in the RNA can be identified. These unusual methods have been developed by the principal investigator and/or her collaborators. Little is known about the properties of major structural features within coding regions of large mRNAs, and our studies are of direct relevance in understanding the role of RNA structure in human diseases such as AIDS and the common cold.

在此提案中，关于RNA调节的两个假设 长距离RNA-RNA相互作用的coliphage基因表达将是 检查：1）Q Beta RNA的5'末端的稳定远程相互作用 抑制病毒成熟蛋白的跨国起始。 （2） 大型结构特征的两个替代构象（CH和ACH）， 中央发夹是法规的结构开关 病毒外套蛋白的跨国起始。 远程RNA 位于基因组RNA的编码区域内的二级结构具有 在基因组和基因组构象的结构研究中被发现 首席研究员的Coliphage Qbeta突变RNA。 他们的 病毒遗传图内的位置表明它们在 跨国启动的调节。 结构研究有 表明来自coliphage qbeta（4217个核苷酸）的基因组RNA可以是 分为一系列很好的结构域 确定预测结构的几个区域 异常塑料。 确定确定的结构域的大小范围 从500到700个核苷酸，包括 QBeta RNA 5'末端的病毒成熟蛋白的翻译。 可变区域包括用于翻译的起始区域 病毒外套和病毒复制酶翻译。 这些站点位于 在中央发夹（1500个核苷酸）的下半部 CH和ACH构象之间的结构重排是 观察到。 在CH构象中，外套的启动区域是免费的，并且 可以合成大量病毒外套蛋白。 在ACH中 构象，外套的启动区域被埋葬在广泛的次要中 结构。 外套合成（以及所有其他病毒蛋白合成）将 关闭。 该开关可以通过病毒和/或细胞介导 蛋白质，可能是通过病毒复制酶。 大型结构开关 类型是新颖的，并且尚未在任何实验中提出过 系统。 拟议的分析将以几个步骤进行。 额外的 需要使用天然和突变体RNA的结构研究来发展 远距离核苷酸水平的可靠结构模型 相互作用在RNA的5'端和CH-的区域 ACH开关。 根据这些研究，将创建突变的RNA 其中建议的远距离相互作用将被破坏。 这些结构变化的效果将在体外和 体内。 这些分析将使用几种异常工具 通过能量点图，全尺寸基因组的潜在结构变化，以及 RNA中的顺式作用结构缺陷的遗传系统可以 被识别。 这些不寻常的方法是由 首席研究员和/或她的合作者。 对 在编码区域内的主要结构特征的特性 大的mRNA，我们的研究与理解 RNA结构在人类疾病（例如艾滋病和普通感冒）中的作用。