The transcriptional transactivator Tat is an essential regulatory protein for HIV replication and AIDS pathogenesis. It binds to the positive transcription elongation factor b (P-TEFb) via the cyclin T1 (cycT1). Together, they bind with high affinity and specificity to the transactivation response (TAR) RNA structure. Cdk9 in P-TEFb then phosphorylates the C-terminal domain (CTD) or RNA polymerase II (RNAPII). This marks the transition from initiation to elongation of HIV transcription. These interactions will be dissected in great detail. Extensive mutageneses will reveal precise binding surfaces on Tat and cycT1. Posttranscriptional modification of cycT1 will be analyzed. These changes could affect the activity and metabolism of P-TEFb in cells. Dominant negative cycT1 proteins and P-TEFb complexes will be created. Additionally, polymorphisms in the human cycT1 will be examined further. They could play an important role in the host response to HIV. The complex between P-TEFb, Tat and TAR will be subjected to X-ray crystallography and NMR. The goal is to obtain the tertiary structure of the complex between P-TEFb, Tat and TAR. To understand Tat transactivation, the mechanism of action of P- TEFb must be examined. Substrates of different P-TEFb complexes besides CTD and SPT5 will be identified biochemically and genetically. They will be tested functionally on different modes of presentation of P-TEFb to the transcription complex. Heterogous DNA and RNA binding proteins will be used. Finally, different cyclins T and K will be inactivated genetically in the mouse? They will reveal how deleterious might be the pharmacological inactivation of the complex between cycT1 and Cdk9 in infected humans. Finally, the interplay between the negative transcription elongation factor (N-TEF), which includes the DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF) and Tat will be dissected on the HIV long terminal repeat (LTR) in vitro and in vivo. First RNA-binding interactions between the lower stem in TAR and RD will be studied. Later DSIF and NELF will be added. Then, all these elements will be combined in a carefully orchestrated temporal fashion, which should reveal the kinetic picture of Tat transactivation. Findings in vitro will be correlated with chromatin immunoprecipitations in cells.
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