RNA POL II POLY-A SITE AND 3' TERMINATION

这是这项建议和我的长期总目标， 实验室了解基因调控通过聚（A）位点使用， 复杂的转录单位对于所有pol II转录单位，3'端 mRNA的形成和转录终止是mRNA 生物合成和转录单位的定义。因为我们是 通过3'端加工定义基因调控，我们也必须 考虑到3'端加工如何影响i.）pol Ⅱ转录 终止（二）剪接位点识别和利用以及iii.） mRNA从细胞核到细胞质的转运。这些步骤中的每一 被认为在战略中发挥着越来越大的作用， 真核基因调控 这项研究计划的具体目标是扩展我们的表征 3'末端形成的分子生物学和生物化学， RNA pol II转录单位的转录终止。 我们 串联poly（A）位点中poly（A）位点使用的表征调节 包含转录单位，以了解poly（A）位点的选择是如何 确定复杂的转录单位。这些研究必然 需要与单个poly（A）位点的功能进行比较。的 给定底物前mRNA的3'加工效率是主要的 这些研究的重点，因为它是一个关键的决定因素，在替代 处理以及转录终止。 我们已经表明，在2000年， 多聚（A）位点是形成“终止”的第一步 感受态”RNA聚合酶II延伸复合物。我们正在扩大我们的 表征3'加工到表征3' 加工导致终止感受态伸长的形成 复杂. 我们正在采取两项并行和互补的战略， 3'加工和转录终止的表征; i.在 体内研究，我们正在使用，以确定功能参数， 调节细胞内的3'端加工和终止 二.体外研究定义了3 '加工的生物化学， 转录终止

It is the long term general objective of this proposal and of my laboratory to understand gene regulation through poly(A) site use in complex transcription units. For all pol II transcription units, 3' end formation and transcription termination are mandatory steps in mRNA biosynthesis and in the definition of the transcription unit. As we are defining gene regulation by 3' end processing, we are necessarily also considering how 3' end processing affects i.) pol II transcription termination ii.) splice site recognition and utilization and iii.) transport of mRNA from the nucleus to the cytoplasm. Each of these steps is recognized as playing an increasingly large role in the strategy of eucaryotic gene regulation. The specific goal of this research plan is to extend our characterization of the molecular biology and biochemistry of 3' end formation and transcription termination in RNA pol II transcription units. We are characterizing regulation of poly(A) site use in tandem poly(A) site containing transcription units to understand how poly(A) site choice is determined for complex transcription units. These studies necessarily require a comparison to function of individual poly(A) sites. The efficiency of 3' processing for a given substrate pre-mRNA is a major focus of these studies since it is a key determinant in alternative processing as well as in transcription termination. We have shown that recognition and presumably cleavage efficiency at the poly(A) site is a required first step in formation of a "termination competent" RNA polymerase II elongation complex. We are extending our characterization of 3' processing to a characterization of how 3' processing causes formation of the termination competent elongation complex. We are pursuing two parallel and complementing strategies for characterization of 3' processing and transcription termination; i. in vivo studies which we are using to identify functional parameters which are operating to regulate 3' processing and termination within the cell ii. in vitro studies defining the biochemistry of 3'processing and transcription termination.