FACTORS INVOLVED IN TRANSCRIPTION BY RNA POLYMERASE II

描述（由申请人提供）：RNA聚合酶II（Pol II）转录的延伸期受到高度调节，并复杂地整合到mRNA生物合成中。为了理解所涉及的因素及其作用机制，提出了有助于理解P-TEFb和TTF 2在控制转录延伸中的作用以及RNA加工在多大程度上与转录功能性耦合的研究。I. P-TEFb在调节起始后不久发生的生产性延伸的关键检查点中起关键作用。P-TEFb的激酶活性反过来通过与含有7SK RNA和HEXIM 1或HEXIM 2的RNP可逆缔合而独特地调节。将使用体外和体内方法检查影响P-TEFb/7SK/HEXIM复合物的形成和解离的参数。P-TEFb在特定基因上和全局上在所有基因上的功能的细节将使用与阵列方法偶联的核连续反应来收集。二.转录在有丝分裂过程中被关闭，TTF 2的RNA Pol I和Pol II终止活性是转录延伸的有丝分裂抑制所必需的。TTF 2在有丝分裂转录抑制中的作用将被进一步研究，并将检查转录偶联修复和间期转录中的潜在作用。还将研究TFIIF和其他延伸因子在转录过程中控制TTF 2活性的潜在作用。三.产生功能性mRNA所需的加工反应部分发生在延伸过程中，并受到转录过程的影响。新开发的测定将用于阐明在转录背景下的人加帽酶和帽甲基转移酶的功能的细节。最后，使用一个新开发的体外系统，我们将确定在何种程度上多聚腺苷酸化功能耦合到转录延长和终止。所描述的大多数研究旨在了解控制基因表达的基本过程，但P-TEFb在HIV基因表达中起着重要作用，可能是艾滋病治疗的靶点。此外，由于一种用于癌症治疗的临床试验药物flavopiridol靶向P-TEFb，因此拟议的研究很重要。

DESCRIPTION (provided by applicant): The elongation phase of transcription by RNA polymerase II (Pol II) is highly regulated and intricately integrated into mRNA biosynthesis. In an effort to understand the factors involved and their mechanisms of action, studies are proposed that will help understand the role of P-TEFb and TTF2 in controlling transcription elongation and to what extent RNA processing is functionally coupled to transcription. I. P-TEFb plays a key role in regulating the critical checkpoint to productive elongation that occurs shortly after initiation. The kinase activity of P-TEFb is in turn uniquely regulated by reversible association with an RNP which contains 7SK RNA and HEXIM1 or HEXIM2. Parameters affecting the formation and dissociation of the P-TEFb/7SK/HEXIM complex will be examined using methods in vitro and in vivo. Details of the function of P-TEFb on specific genes and globally over all genes will be gathered using nuclear run-on reactions coupled to array methods. II. Transcription is shut off during mitosis and the RNA Pol I and Pol II termination activity of TTF2 is required for mitotic repression of transcription elongation. The role of TTF2 in mitotic transcription repression will be further studied and potential roles in transcription coupled repair, and interphase transcription will be examined. Also the potential role of TFIIF and other elongation factors in controlling TTF2 activity during transcription will be investigated. III. The processing reactions needed to generate functional mRNAs occur in part during elongation and are influenced by the transcription process. Newly developed assays will be used to elucidate the details of the function of the human capping enzyme and the cap methyl transferase in the context of transcription. Finally, using a newly developed in vitro system we will determine the extent to which polyadenylation is functionally coupled to transcription elongation and termination. Most of the studies described are aimed at understanding basic processes controlling gene expression, but P-TEFb plays a major role in HIV gene expression and may be a target for AIDS therapy. Also because a drug in clinical trials for cancer therapy, flavopiridol, targets P-TEFb, the proposed studies are important.