Structural understanding of 7SK-snRNP mediated transcriptional regulation

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Abstract During transcription of many cellular and viral mRNAs, RNA Polymerase II is inhibited by negative elongation factors and requires the positive elongation factor, pTEFb, to release this stalled state. The binding of the 7SK small nuclear RNA to the HEXIM adapter protein as a part of the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex normally keeps pTEFb sequestered within the nucleus and this interaction must be overcome for productive transcriptional elongation. The most well-studied example of the regulatory mechanisms that underlie transcriptional elongation is the HIV Tat system. Briefly, in order to release the stalled state of its transcript, HIV has evolved the viral Tat protein, which binds 7SK and displaces HEXIM to hijack pTEFb. On the other hand, some cellular factors, like BRD4, achieve the same outcome without displacing HEXIM. This proposal aims to gain a mechanistic understanding of the structural state presented to all transcriptional regulators encountering an HEXIM-driven inhibitory 7SK snRNP complex, and how capture of pTEFb is achieved by the specialized HIV Tat transcriptional factor. The aims will be: (#1) to understand if the basic mechanisms are maintained in HEXIM1 and 2 and if diverse positive transcriptional regulators capitalize on the mechanism, (#2) to determine the structures of the individual RNA domains present in 7SK- snRNA and (#3) to solve the structures of both HEXIM-bound and Tat-bound complexes.