CYCLIN T AND HIV1 TAT TRANSACTIVATION

我们最近分离到一种新的C型细胞周期蛋白（称为细胞周期蛋白T），它通过其激活结构域直接与HIV-1达特相互作用。 细胞周期蛋白T（CycT）是CDK 9（PITALRE）的主要细胞周期蛋白伴侣，CDK 9是正作用转录延伸因子复合物P-TEFb的催化亚基。 我们确定达特与CycT的相互作用显著增强了其与TAR RNA的结合，并赋予了TAR环中不被游离（未复合的）达特蛋白识别的关键序列的需要。 细胞周期蛋白T与达特相互作用的结构-功能分析。 在这里，我们建议表征达特和CycT之间的相互作用在分子上的细节，并确定其他因素存在于Tat相关激酶（TAK）/P-TEFb复合物，可能需要调节转录延伸的达特。 在特定目标1中，我们将使用定点诱变和UV交联技术鉴定CycT和达特中相互作用和与Tar RNA结合所必需的残基。 在特定目标2中，我们将分析野生型和突变型CycT蛋白支持基础和Tat介导的体内反式激活的能力，以及在hCycT免疫耗竭或用反式显性达特（1-48）蛋白阻断的无细胞转录反应中的能力。 我们还将确定达特活性是否需要额外的P-TEFb组分。 我们最近克隆了鼠CycT蛋白（mCycT），发现它不能通过TAR支持达特反式激活。 在特定目标3中，我们将描述mCycT中的缺陷，并确定恢复体内达特反式激活所需的最小变化。 最后，在具体目标4和5中，我们提出克隆和表征两种新的蛋白质，我们发现它们单独或与TAR RNA结合时与核提取物中的TAK/P-TEFb紧密相关。总之，这些研究将为HIV达特反式激活机制提供重要的新信息。

We have recently isolated a novel C-type cyclin (called Cyclin T) which interacts directly with HIV-1 Tat through its activation domain. Cyclin T (CycT) is a predominant cyclin partner for CDK9 (PITALRE), the catalytic subunit of the positive-acting transcription elongation factor complex, P-TEFb. We determined that the interaction of Tat with CycT dramatically enhances its binding to TAR RNA, and confers a requirement for critical sequences in the loop of TAR which are not recognized by the free (uncomplexed) Tat protein. Structure-function analysis of the cyclin T:Tat interaction. Here we propose to characterize the interaction between Tat and CycT in molecular detail, and to identify other factors present in the Tat-associated kinase (TAK)/P-TEFb complex that may be required for the regulation of transcription elongation by Tat. In Specific Aim 1 we will identify residues within CycT and in Tat that are necessary for their interaction and for binding to Tar RNA using site-directed mutagenesis and UV cross-linking techniques. In Specific Aim 2, we will analyze the ability of wild-type and mutant CycT proteins to support basal and Tat-mediated transactivation in vivo and in cell-free transcription reactions that have been immunodepleted of hCycT, or blocked with trans-dominant Tat (1-48) protein. We will also determine whether additional P-TEFb components are needed for Tat activity. We have recently cloned the murine CycT protein (mCycT) and find that it is unable to support Tat transactivation through TAR. In Specific Aim 3, we will characterize the defect in mCycT and identify the minimal changes needed to restore Tat transactivation in vivo. Finally, in Specific Aims 4 and 5 we propose to clone and characterize two novel proteins which we find to be tightly associated with TAK/P- TEFb in nuclear extracts, either alone or when bound to TAR RNA. Taken together, these studies will provide important new information on the mechanism of HIV Tat transactivation.