Structure of the Cyclin T binding domain of Hexim1 and molecular basis for its recognition of P-TEFb

Abstract

Hexim1 is a cellular protein that associates with the positive transcription elongation factor b (P-TEFb) to regulate RNA polymerase II elongation of nascent mRNA transcripts. It directly binds to Cyclin T1 of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest of transcription elongation. Here, we report the solution structure of the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel coiled-coil homodimer composed of two segments and a preceding alpha helix that folds back onto the first coiled-coil unit. NMR titration, fluorescence, and immunoprecipitation experiments revealed the binding interface to Cyclin T1, which covers a large surface on the first coiled-coil segment. Electrostatic interactions between an acidic patch on Hexim1 and positively charged residues of Cyclin T1 drive the complex formation that is confirmed by mutagenesis data on Hexim1 mediated transcription regulation in cells. Thus, our studies provide structural insights how Hexim1 recognizes the Cyclin T1 subunit of P-TEFb, which is a key step toward the regulation of transcription elongation.Cyclin T1 ͉ NMR spectroscopy ͉ transcription elongation T ranscriptional elongation by RNA polymerase II (pol II) is a highly regulated process that generates full-length RNA transcripts and coordinates transcription with pre-mRNA processing (1). The positive transcription elongation factor b (P-TEFb) is a key regulator for the transition from abortive to productive elongation of mature mRNA molecules (2). The core form of P-TEFb is a heterodimer between the cyclin-dependent kinase 9 (Cdk9) and its regulatory subunit Cyclin T1 (CycT1) or the minor forms T2 or K. Transcription activation by P-TEFb depends on the kinase activitiy of Cdk9, which phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA pol II to stimulate the processivity of elongation (3). Initially, P-TEFb has been found to play a critical role in stimulating the transcription of HIV-1 genes, where the viral transactivator protein Tat recruits P-TEFb to stalled RNA pol II molecules (2). At present, P-TEFb is considered a global transcriptional elongation factor important for the expression of most RNA pol II-regulated genes (4).Recently, it has been found that P-TEFb itself is subject to a tight regulation, because approximately half of nuclear P-TEFb in HeLa cells is sequestered in an inactive state. This inactive complex is composed of the Cdk9/CycT1 heterodimer bound to the abundant small nuclear RNA 7SK and the regulatory protein Hexim1 (5-10). Whereas 7SK is supposed to serve as a molecular scaffold to mediate the interaction of Hexim1 with P-TEFb, Hexim1 binds directly to the cyclin box repeats of CycT1 and inhibits the kinase activity of Cdk9. The other half of P-TEFb heterodimers is catalytically active and binds the bromodomain protein, Brd4 (11, 12), which has high affinity for the acetylated tails of core histones H3 and H4 and can bind the Mediator complex.Hexim1 was initially identified as a protein whose expression is induced in...

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…6b), although the TBD was shown before to interact with CycT1 at a site required in the Cdk2-CycA complex for substrate recognition 27 . Further N-terminal elongations of the TBD revealed a marked increase in the inhibitory potential of a construct starting at position 194 compared with position 207, suggesting that residues 194-206 are necessary for the inhibitory function of Hexim1 (Fig.…”

Structures of the Dual Bromodomains of the P-TEFb-activating Protein Brd4 at Atomic Resolution

“…Crystal structures of the Cdk9/CycT1 heterodimer and the activating CycT1/Tat/TAR complex gave first insights into the molecular basis of P-TEFb regulation (41,42). Because the lentiviral Tat protein was shown to displace the cellular P-TEFb regulator Hexim1 from a mutual binding site on CycT1, Hexim1 is supposed to bind to a similar surface on the first cyclin box repeat of CycT1 (28,43,44). In contrast, Brd4 bromodomains were initially described to interact with a region in CycT1 that is located C-terminal to the cyclin box repeat, whereas a later report showed the far C terminus of Brd4 to be required and sufficient for P-TEFb activation (6 -8).…”

7SK RNA, a non-coding RNA regulating P-TEFb, a general transcription factor

“…63 HEXIM1/2 proteins form stable dimers through a bipartite coiled-coil leucine zipper in their C-terminal domain. 59,64 Thus, the BR1/AR1 interaction might be inter-or intra-molecular. The resulting conformation might mask or impair the surface interacting with P-TEFb.…”