REGULATION OF TRANSCRIPTIONAL ELONGATION BY HIV1 TAT

转录延伸的控制已经被认为是一种 基因调控中的重要一步，但调控机制， 通过RNA聚合酶II延伸的效率尚未被 广泛研究。拟议研究的目标是 用HIV-1达特作为一种免疫调节剂， 模型系统达特通过以下方式刺激伸长的效率： 识别反式作用应答（TAR）RNA元件， 在新生病毒转录物的5 ′端。达特反式激活 需要特定的细胞辅因子。申请人最近 分离并克隆了一种细胞辅因子Tat-SFI， 是伸长的达特活化所特别需要的，并且是 相关的linase的底物。Tat-SFI与 EWS和FUS/TLS，它们是一类新的 具有RNA识别基序的推定转录因子， 常与肉瘤有关。初步研究表明 达特可能通过募集一种新的蛋白质来刺激伸长的效率， 含有Tat-SFI及其激酶的预形成复合物对HIV的作用 启动子和通过增强延伸因子的作用， Elongin（Sill）。这项建议旨在进一步剖析其职能， Tat-SFI在达特激活中的作用 达特、塔特-SFI、达特-SFI之间的各种相互作用的特异性。 SFI激酶和TAR，并通过绘制和氨基酸 Tat-SFI活性的关键残基。一个重要的目标是 评估Tat-SFI有趣的功能意义 在达特活化中的磷酸化，并分离、克隆和 表征其激酶。还将作出重大努力， 研究达特和达特SFI的机制 刺激延伸的效率和鉴定 Tat刺激的延伸聚合酶复合物的组分。 最后，达特和细胞伸长之间的关系 聚合酶激活中的因子如Elongin和TFIIS 将研究持续性。进一步分析达特激活 延长的效率和细胞辅因子的作用 在这个过程中可能会揭示基因的一般机制， 伸长阶段的调节。

Control of transcriptional elongation has been recognized as an important step in gene regulation, but mechanisms regulating the efficiency of elongation by RNA polymerase II have not been extensively studied. The goal of the proposed research is to elucidate mechanisms regulating elongation using HIV-l Tat as a model system. Tat stimulates the efficiency of elongation by recognizing the trans-acting-response (TAR) RNA element located at the 5' end of the nascent viral transcript. Tat trans-activation requires specific cellular cofactors. The applicant has recently isolated and cloned a cellular cofactor, Tat-SFI, which is specifically required for Tat activation of elongation and is a substrate of an associated linase. Tat-SFI is distantly related to EWS and FUS/TLS, which are members of a novel class of putative transcription factors with RNA recognition motifs and are frequently associated with sarcomas. Preliminary studies suggest that Tat may stimulate the efficiency of elongation by recruiting a preformed complex containing Tat-SFI and its kinase to the HIV promoter and by enhancing the effect of an elongation factor, Elongin (Sill). This proposal seeks to further dissect the function of Tat-SFI in Tat activation through analyzing the sequence specificity of the various interactions among Tat, Tat-SFI, the Tat- SFI kinase, and TAR, and through mapping the and amino acid residues critical for Tat-SFI activity. One important objective is to assess the intriguing functional significance of Tat-SFI phosphorylation in Tat activation and to isolate, clone, and characterize its kinase. A significant effort will also be devoted to the investigation of the mechanisms by which Tat and Tat SFI stimulate the efficiency of elongation and the identification of components of a Tat-stimulated elongating polymerase complex. Finally, the relationship between Tat and cellular elongation factors such as Elongin and TFIIS in activation of polymerase processivity will be studied. Further analysis of Tat activation of the efficiency of elongation and the role of the cellular cofactors in this process will likely reveal general mechanisms of gene regulation at the stage of elongation.