Mechanism of Initiation by RNA polymerase II

RNA 聚合酶 II 的引发机制

• 批准号: 6898774
• 负责人: JAY D GRALLA
• 金额: $ 29.51万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6898774
• 关键词: DNA directed RNA polymerase; DNA repair; adenosine triphosphate; adenosinetriphosphatase; deafness; dwarfism; enzyme activity; enzyme mechanism; gel mobility shift assay; gene mutation; genetic promoter element; genetic transcription; human genetic material tag; messenger RNA; molecular pathology; nucleic acid sequence; protein protein interaction; protein structure; protein structure function; retina degeneration; skin disorder; transcription factor; xeroderma pigmentosum; yeasts

DESCRIPTION (provided by applicant): This proposal is aimed at learning the roles of the XPB and XPD subunits of the multi-protein factor TFIIH. TFIIH is essential for transcription of RNA, repair of DNA and control of cellular mitosis. XPB is essential to both transcription and repair and XPD is used for repair and mitotic control. 3 human diseases track to mutations in the XPB and XPD subunits, XP (xeroderma pigmentosum), CS (Cockaynes syndrome) and TTD (Trichothiodystrophy). TTD and CS are caused in significant part by transcription defects. There are major gaps in our knowledge of how these proteins function and how the mutated proteins contribute to CS and TTD. This proposal aims to fill those gaps. One goal is to determine how XPB leads to DNA opening during transcription. Binding and helicase assays using purified human components will be used to see how XPB and TFIIH cooperate with both basal transcription factors and activators to trigger appropriate promoter opening and transcription. In addition, mutations will be introduced into XPB in both human and fission yeast, and the effect on transcription and DNA repair determined within crude cellular extracts. Some of these mutations will mimic alleles that cause XPB diseases of transcription. A similar but less extensive analysis will be done with mutant XPD alleles. The role of other XP factors in facilitating communication between the transcription and DNA repair pathways will also be explored. These experiments are directed towards understanding whether there is an active mechanism for distributing TFIIH between transcription and DNA repair complexes. They will evaluate the roles of DNA repair factors known to bind transcription components by adding them to transcription extracts. How they interact with isolated transcription factors will also be explores to learn the extent of active communication between these 2 critical cellular processes.

描述（由申请人提供）：该建议旨在学习多蛋白质因子TFIIH的XPB和XPD亚基的作用。 TFIIH对于RNA的转录，DNA的修复和控制细胞有丝分裂是必不可少的。 XPB对于转录和修复至关重要，XPD用于修复和有丝分裂控制。 3人类疾病在XPB和XPD亚基中的突变，XP（Xeroderma cipmentosum），CS（Cockaynes综合征）和TTD（Trichothiododystrophy）。 TTD和CS是由转录缺陷引起的。我们了解这些蛋白质如何功能以及突变蛋白如何对CS和TTD贡献的主要差距。 该建议旨在填补这些空白。一个目标是确定XPB在转录过程中如何导致DNA打开。使用纯化的人组件的结合和解旋酶测定将用于查看XPB和TFIIH如何与基础转录因子和激活剂合作，以触发适当的启动子开放和转录。另外，将在人和裂变酵母中引入突变，以及对粗细细胞提取物中确定的转录和DNA修复的影响。其中一些突变会模仿引起XPB转录疾病的等位基因。突变XPD等位基因将进行类似但不太广泛的分析。 还将探讨其他XP因素在促进转录和DNA修复途径之间通信中的作用。这些实验是针对了解在转录和DNA修复复合物之间分布TFIIH的主动机制的。他们将通过将其添加到转录提取物中来评估已知结合转录成分的DNA修复因子的作用。 它们如何与孤立的转录因子相互作用也将进行探索，以了解这两个关键细胞过程之间的主动通信程度。