STRUCTURAL STUDIES OF BACILLUS SUBTILIS SIGMA FACTORS

遗传物质的协调和及时表达是一个关键 所有生物体的发育、生长和适应方面。 相反地， 基因的不当表达会导致癌症和许多其他疾病， 疾病过程。 在真细菌中，将DNA代码精确复制到 RNA（转录）需要一个σ因子家族， RNA聚合酶核心亚基，以允许识别 基因（启动子）。 sigma因子的作用被认为包括 在转录前两条DNA链的专性分离 启动事件。 假设sigma因子作为DNA解链蛋白， 高亲和力，单链DNA结合位点的起始点， 转录将被测试。 这个结合位点的位置 从sigma因子和a之间的氨基酸序列比较预测 真核单链核酸结合蛋白家族 并且被认为包括保守的芳香族氨基酸 在细菌进化中。 我们将从基因上改变这些残留物 并确定这些改变对生物化学功能的影响 sigma因素。 我们的研究将集中在一个主要的西格玛 枯草芽孢杆菌sigmaA蛋白，一种代表性的替代物， sigma因子，即B。枯草杆菌sigmaD蛋白。 sigmaA蛋白将 提供了一个生化系统的影响分析网站- 对sigma因子结构和功能的直接遗传变化。 一 互补遗传系统可用于识别功能改变 σ D等位基因 作为确定地点的另一种方法， sigma因子：DNA接口，我们将利用观察结果， 当RNA聚合时，σ因子与启动子DNA共价交联， 将聚合酶：启动子复合物暴露于紫外光。 的 参与形成这种交联的氨基酸的鉴定将 帮助定位与DNA接触的σ因子区域。 这些和相关实验将允许特定地点的机制 待阐明的RNA聚合酶的DNA解链。 预计各国 这些研究将提供对转录激活因子作用的深入了解， 蛋白质促进转录起始过程，甚至可能 揭示了真核转录催化的类似反应， 起始蛋白和DNA复制起始蛋白。

The coordinated and timely expression of the genetic material is a crucial facet of development, growth and adaptation in all organisms. Conversely, the misappropriate expression of genes contributes to cancer and many other disease processes. In eubacteria, the precise copying of the DNA code into RNA (transcription) requires one a family of sigma factors that bind to the RNA polymerase core-subunits to allow recognition of the start points of genes (promoters). The role of sigma factors is thought to include the obligate separation of the two DNA strands prior to the transcription initiation event. The hypothesis that sigma factors act as DNA-melting proteins by providing a high affinity, single-stranded DNA binding site at the start point of transcription will be tested. This location of this binding site has been predicted from amino acid sequence comparisons between sigma factors and a family of eukaryotic single-stranded nucleic acid binding proteins (RNPs) and is thought to include aromatic amino acids that have been conserved throughout bacterial evolution. We will alter these residues genetically and determine the affects of these alterations on the biochemical functions of sigma factors. Our studies will focus on one of the major sigma factors, the Bacillus subtilis sigmaA protein, a representative alternative sigma factor, the B. subtilis sigmaD protein. The sigmaA protein will provide a biochemical system for the analysis of the affects of site- directed genetic changes on sigma factor structure and function. A complementary genetic system is available to identify altered-function alleles of sigmaD. As an additional approach to identify the location of the sigma factor: DNA interface we will make use of the observation that sigma factors become covalently crosslinked to promoter DNA when RNA polymerase:promoter complexes are exposed to ultraviolet light. The identification of the amino acids involved in forming this crosslink will help localize the regions of sigma factor in contact with DNA. These and related experiments will allow the mechanism of site-specific DNA-melting by RNA polymerase to be elucidated. It is anticipated that these studies will provide insight into the role of transcription activator proteins in facilitating the transcription initiation process and may even shed light on the analogous reactions catalyzed by eukaryotic transcription initiation proteins and by DNA replication initiation proteins.