Role of protein phosphorylation in Myulating RNA polymerase II transcription

1. ERCC3在gal4 - vp16转录激活中的作用在基础转录中，ERCC3解旋酶在启动子打开和启动子逃逸中起作用。我们测试了在GAL4-VP16的转录激活过程中ERCC3解旋酶在哪个步骤起作用。TFIIH突变体的作用首先使用预熔模板进行测试，发现TFIIH在激活转录的启动子逃逸过程中起作用。这种效果是特定于激活的。利用各种突变模板，我们发现激活因子在转录的早期阶段起作用。这种效果需要辅激活剂PC4。RNA聚合酶II在转录的早期阶段是不稳定的，可能导致转录中止。通过使用ATP类似物，我们发现当ERCC3解旋酶活性被阻断时，RNA聚合酶II可能会从模板上分离。总的来说，转录激活涉及转录机制的招募和启动子逃逸的增强。启动子逃逸的速率常数在激活转录中不受刺激。TFIIH在转录偶联盖帽反应中的作用与mizumoto博士合作，我们开发了一个系统，其中转录和盖帽反应可以一起发生。该系统包括除RNA聚合酶II外的重组因子。在该体系中，盖帽反应与转录起始同时发生。帽盖的分子分析及其与转录起始的关系正在进行中。

1. Role of ERCC3 in transcriptional activation by GAL4-VP16In basal transcription, ERCC3 helicase acts during promoter opening and promoter escape. We tested at which step ERCC3 helicase acts during transcriptional activation by GAL4-VP16. The effect of TFIIH mutants were first tested using premelted templates, and revealed that TFIIH functions during promoter escape in activated transcription. The effect is specific to activation. Using the various mutant templates, we found that activators act during a very early stage of transcription. The effect requires coactivator PC4. RNA polymerase II is unstable during the early phase of transcription and may abort transcription. With the use of ATP analogs, we found that RNA polymerase II may detach from the template when ERCC3 helicase activity is blocked. Taken together, transcriptional activation involves the enhancement of recruitment of transcriptional machinery and promoter escape. Rate constant of promtoer escape is not stimulated in activated transcription.2. Role of TFIIH in transcription-coupled capping reactionsIn collaborated with Dr.Mizumoto, we have developed a system in which transcription and capping reactions can occur together. The system includes recombinant factors except RNA polymerase II.In this system, capping reaction occurs simultaneously with transcription initiation. The molecular analyses of capping and its relation to transcription initiation are in progress.