CONTROL OF TRNA GENE EXPRESSION IN BOMBYX MORI

本研究的目的是探讨 通过家蚕RNA聚合酶III体外转录。 我们的大部分努力 将集中在一个新的转录成分，因子X，我们的 实验室最近被隔离。 这个因素是一个重要的组成部分， 转录真核生物tRNA和5S基因的机制。 一般 假设这种机器完全由多肽组成。 的 因子X的一个显著特征是它似乎是一种RNA，而不是 蛋白质。具有转录活性的RNA的发现是 史无前例 X因子的发现来自于我们的系统研究， tRNA和5S RNA转录所需组分的分级分离 体外 它与我们以前解决的组件不同。 实验室和其他：聚合酶III，和转录因子A，B， C和d因子X看起来是一种核酸，因为它能抵抗 热、去污剂、苯酚和蛋白酶，但对核酸酶敏感。 它 似乎是一种RNA，因为它对DNA酶有抗性，但对 RNA酶和碱。 本建议的具体目标是：（1）确定关键的 大tRNA（Ala）C启动子的功能所必需的片段， (2)为了纯化因子X以及其他蚕转录因子， 为了明确地建立他们的身份，（3）分配 对因子X和其他转录因子的特异性功能，通过（a） 确定它们是否参与因子模板和因子因子 （B）确定细胞周期的哪个阶段需要因子 （c）构建因子X的突变体形式以测试特异性 假设其功能。

The aim of the proposed work is to investigate the mechanism of transcription by silkworm RNA polymerase III in vitro. Much of our effort will focus on a novel transcription component, Factor X, that our laboratory has recently isolated. This factor is an essential part of the machinery that transcribes eukaryotic tRNA and 5S genes. It is generally assumed that such machinery is composed entirely of polypeptides. The remarkable feature of Factor X is that it appears to be an RNA rather than a protein. The finding of an RNA with transcription activity is unprecedented. The discovery of Factor X came from our systematic fractionation of the components required for tRNA and 5S RNA transcription in vitro. It is distinct from the components previously resolved by our laboratory and others: polymerase III, and the transcription factors A, B, C, and D. Factor X appears to be a nucleic acid because it is resistant to heat, detergent, phenol, and protease, but sensitive to nuclease. It appears to be an RNA because it is resistant to DNAse, but sensitive to RNAse and alkali. The specific aims of this proposal are: (1) to identify the critical segments of the large tRNA(Ala)C promoter that are essential for function, (2) to purify Factor X, as well as the other silkworm transcription factors in order to establish their identities unambiguously, (3) to assign specific functions to Factor X and the other transcription factors by (a) determining whether they participate in factor-template and factor-factor interactions, (b) determining which phase of the cell cycle requires Factor X, and (c) constructing mutant versions of Factor X to test specific hypotheses for its function.