TFIIH Inhibits CDK9 Phosphorylation during Human Immunodeficiency Virus Type 1 Transcription

Abstract

Tat stimulates human immunodeficiency virus, type 1 (HIV-1), transcription elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to the TAR RNA structure. It has been demonstrated further that CDK9 phosphorylation is required for high affinity binding of Tat/P-TEFb to the TAR RNA structure and that the state of P-TEFb phosphorylation may regulate Tat transactivation. We now demonstrate that CDK9 phosphorylation is uniquely regulated in the HIV-1 preinitiation and elongation complexes. The presence of TFIIH in the HIV-1 preinitiation complex inhibits CDK9 phosphorylation. As TFIIH is released from the elongation complex between ؉14 and ؉36, CDK9 phosphorylation is observed. In contrast to the activity in the "soluble" complex, phosphorylation of CDK9 is increased by the presence of Tat in the transcription complexes. Consistent with these observations, we have demonstrated that purified TFIIH directly inhibits CDK9 autophosphorylation. By using recombinant TFIIH subcomplexes, our results suggest that the XPB subunit of TFIIH is responsible for this inhibition of CDK9 phosphorylation. Interestingly, our results further suggest that the phosphorylated form of CDK9 is the active kinase for RNA polymerase II carboxyl-terminal domain phosphorylation.

TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb

“…A well documented example, with a strikingly similar pattern of interactions, is the HIV transactivator protein Tat. Like TAF7, Tat (i) binds TAF1 and inhibits its acetyl transferase activity (23), (ii) binds P-TEFb and activates its autophosphorylation (34), and (iii) modulates the phosphorylation of RNAP CTD by TFIIH and P-TEFb (35). Although there is no structural similarity between these two proteins, the remarkable parallels between TAF7 and Tat function suggest that Tat has subverted the cellular processes normally regulated by TAF7.…”

Cross-talk Among RNA Polymerase II Kinases Modulates C-terminal Domain Phosphorylation

“…An important corollary to this model is that a cascade of phosphorylations regulates CTD phosphorylation, transcription, and co-transcriptional nuclear processes: CDK7 regulates BRD4, which in turn regulates PTEFb. It is noteworthy that CDK7 does not interact with CDK9 (20) although XPB, a component of TFIIH separate from CDK7/CAK, inhibits CDK9 autophosphorylation but not its CTD kinase activity (27). Layered upon the direct modifications that regulate CTD kinase activities are the interactions with TAF7 that provide indirect regulation.…”

The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA

“…After TFIIH is released from TECs between ϩ14 and ϩ36, CDK9 is activated and induces Tat͞TAR-dependent phosphorylation of the RNAP II CTD (13). In vivo crosslinking experiments in budding yeast showed that recruitment of CEs to the 5Ј ends of nascent transcripts requires Kin 28, the kinase subunit of TFIIH that phosphorylates Ser-5 (27,28).…”

“…TFIIH and P-TEFb are two principal CTD kinases during HIV-1 transcription (12,13). In the HIV-1 PICs, TFIIH phosphorylates Ser-5 of RNAP II CTD whereas P-TEFb phos- Incorporation of 32 P into RNAP II was assessed by SDS͞PAGE followed by autoradiography (Upper).…”

“…RNAP II CTD is phosphorylated at Ser-5 by TFIIH during transcription initiation through the promoter clearance stage and changes to Ser-2 phosphorylation when the polymerase is associated with the coding region (11). However, RNAP II CTD Ser-5 phosphorylation is sustained by Tat͞transactivation response (TAR)-induced P-TEFb following release of TFIIH from HIV-1 transcription elongation complexes (TECs) at the promoter clearance stage (12,13). Chromatin immunoprecipitation (ChIP) assays show an accumulation of RNAP II phosphorylated at Ser-5 in the promoter proximal regions of a number of genes (14,15), suggesting that the role of RNAP II CTD phosphorylation in RNA processing is more than mere anchoring of transcription factors.…”