Mechanisms regulating HIV-1 gene expression

描述(申请人提供)：Tat蛋白是一种病毒反式激活剂 即通过增加RNA的能力来激活HIV-1基因的表达 聚合酶II能够有效地进行转录延伸。增加了 RNA聚合酶II的加工性可以通过改变磷酸化来调节 RNA聚合酶II的状态及其与特定伸长率的关联 转录复合体中的因子。研究表明，TAT的激活 HIV-1转录延长是由其与TAR RNA结合介导的 结合由细胞周期蛋白Ti和CDK9组成的pTEF-b激酶复合体 提高伸长因子SPT4/SPT5的活性。这个机制是通过 其中TAT/pTEF-b复合体刺激RNA聚合酶IL在 是否与SPT4/SPT5结合仍有待确定。 在这项提案中，我们利用了许多方法，以便更好地 了解TAT促进HIV-1转录的机制 伸长率。我们使用生化分馏技术鉴定了两种新的 与细胞周期蛋白Ti和SPT5结合的因子。此外，我们还发现CDK9 细胞周期中的变化会改变激酶的活性，提示存在联系 调节细胞周期和细胞基因表达之间的关系。这个 CDK9激酶活性的变化与磷酸化程度的增加相关 RNA聚合酶II和SPT5的状态。最后，我们使用了Rina 干扰--一种干扰特定基因功能的有效方法 在低等物种中，开始定义体内转录途径 参与TAT的激活。 这项提案的目标是阐明以下具体步骤 参与TAT激活HIV-1基因的表达。具体目标是： (1)鉴定细胞蛋白JBP-1和PP2C的功能 与SPT4/SPT5和细胞周期蛋白T1/CDK9相关。(2)了解机制 调节细胞周期中CDK9激酶活性的变化。(3)至 检测CDK9/细胞周期蛋白在SPT5中的磷酸化对其调控作用 刺激转录延长的能力。(4)利用RNA干扰 定义参与调控的转录途径 TAT激活。这些研究将解决涉及到的不同机制 调节TAT激活HIV-1基因表达的能力。

DESCRIPTION (provided by applicant): The Tat protein is a viral transactivator that is activates HIV-1 gene expression by increasing the ability of RNA polymerase II to efficiently undergo transcriptional elongation. Increases in RNA polymerase II processivity can be modulated by altering the phosphorylation state of RNA polymerase II and by the association of specific elongation factors with the transcription complex. Studies indicate that Tat-activation of HIV- 1 transcription elongation is mediated by its binding to TAR RNA in conjunction to the pTEF-b kinase complex comprised of cyclin Ti and CDK9 to increase the activity of the elongation factors SPT4/SPT5. The mechanism by which the Tat/pTEF-b complex stimulates RNA polymerase IL processivity in conjunction with SPT4/SPT5 remains to be determined. In this proposal, we utilize a number of approaches in order to better understand the mechanism by which Tat increases HIV- 1 transcriptional elongation. We have used biochemical fractionation to identify two novel factors that bind to cyclin Ti and SPT5. In addition, we have found that CDK9 kinase activity is altered by changes in the cell cycle suggesting links between the regulation of the cell cycle and cellular gene expression. The changes in CDK9 kinase activity correlate with increases in the phosphorylation state of both RNA polymerase II and SPT5. Finally, we have used RINA interference, a well characterized method to interrupt specific gene function in lower species, to begin to define the in vivo transcriptional pathways involved in Tat activation. The objectives of this proposal are to elucidate the specific steps that are involved in Tat-activation of HIV- 1 gene expression. The specific aims are: (1) To characterize the function of cellular proteins JBP 1 and PP2C that are associated with SPT4/SPT5 and cyclin T1/CDK9. (2) To understand the mechanisms regulating the changes in CDK9 kinase activity during the cell cycle. (3) To determine the role of CDK9/ cyclin Ti phosphorylation of SPT5 on regulating its ability to stimulate transcriptional elongation. (4) To use RNA interference to define the transcriptional pathways that are involved in regulating Tat-activation. These studies will address the different mechanisms involved in regulating the ability of Tat to activate HIV- 1 gene expression.