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.
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