Transcription-dependent Association of Multiple Positive Transcription Elongation Factor Units to a HEXIM Multimer

Cyprien Dulac,Annemieke A Michels,Alessandro Fraldi,François Bonnet,Van Trung Nguyen,Giuliana Napolitano,Luigi Lania,Olivier Bensaude

doi:10.1074/jbc.m502471200

Abstract

The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homo- and hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb.HEXIM1.7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb.HEXIM1.7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.

Highlights

The positive transcription elongation factor (P-TEFb)[1] comprises a protein kinase, CDK9, and a Cyclin T1, T2, or K (1, 2)
Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA
Characterization of FLAG-HEXIM2—To investigate HEXIM2 properties, its cDNA was fused to a FLAG epitope and expressed in HeLa cells

Summary

Introduction

The positive transcription elongation factor (P-TEFb)[1] comprises a protein kinase, CDK9, and a Cyclin T1, T2, or K (1, 2). It is required for transcription elongation of most class II genes. Small nuclear RNA genes do not have a strong requirement for P-TEFb activity to elongate, but rather to terminate, transcription properly (3, 4). The active form consists of “core” P-TEFb, CDK9 and Cyclin T1 or T2. The inactive form consists of CDK9, Cyclin T1 or T2, MAQ1/HEXIM1, and 7SK RNA (5– 8). HEXIM1 and HEXIM2 form stable homo- and hetero-oligomers. These findings were extended, mapping domains involved in HEXIM/HEXIM interactions. It was demonstrated that multiple P-TEFb units bind to a HEXIM1 multimer

Methods

Results

Conclusion