RNA POL II POLY-A SITE AND 3' TERMINATION

这是这项建议和我的 实验室通过Poly(A)位点使用了解基因调控 复杂的转录单位。对于所有POL II转录单位，3‘端 形成和转录终止是信使核糖核酸的必需步骤 在生物合成和转录单位的定义中。就像我们一样 通过3‘端加工来定义基因调控，我们也必然 考虑到3‘端处理如何影响i。)POL II转录 终止协议二。)3.剪接位点的识别和利用。) 信使核糖核酸从细胞核到细胞质的运输。其中的每一步 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特征 3‘末端形成的分子生物学和生物化学 RNA polII转录单位的转录终止。我们是 串联多聚(A)位点使用规律的表征 包含转录单位以了解Poly(A)位点选择是如何 为复杂转录单位确定的。这些研究是必要的 需要与单个Poly(A)位点的功能进行比较。这个 对于给定的底物前-mRNA的3‘处理效率是主要的 这些研究的重点，因为它是替代方案的关键决定因素 处理以及在转录终止中。 我们已经证明，识别和可能的切割效率在 Poly(A)站点是形成“终止”的必要的第一步 有能力的“RNA聚合酶II延伸复合体。我们正在扩展我们的 3‘加工的表征到3’如何加工的表征 加工导致终端合格伸长率的形成 很复杂。 我们正在推行两个平行和相辅相成的战略，以 3‘加工和转录终止的特征；I.in 我们用来确定功能参数的活体研究 正在运行以调节细胞内的3‘处理和终止 二、定义3‘加工和3’的生物化学的体外研究 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.