The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.
The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNArecognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152-155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181-359). Consistently, point mutations in an evolutionarily conserved motif (aa 202-205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.
Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclindependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system. Phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) is a critical step required for transcription elongation (7) and for recruitment of the machinery involved in pre-mRNA maturation (3,26,46). The CTD is unphosphorylated when RNAP II assembles onto promoters (RNAP IIA). A class of negative transcription factors including the 5,6-dichlorozo-1--D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and the negative elongation factor causes transcriptional arrest shortly after initiation, during which the polymerase may fall off (60). To release this block, the CTD must be phosphorylated (RNAP IIO) by positive transcription elongation factor b (P-TEFb), a protein complex that comprises cyclin-dependent kinase 9 (CDK9) and a cyclin (T1 or T2) (45). P-TEFb kinase activity is required for transcription of most class II genes (6).The human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter uses a unique mechanism: the level of proviral DNA transcription is determined by recruitment of P-TEFb to the TAR (transactivation response) element, an RNA stem-loop structure that forms at the 5Ј end of the viral transcript (4,38,59,66). The viral genome encodes a very potent transactivator of its own transcription, the Tat protein.The formation of a quaternary complex among CDK9, cyclin T1, Tat, and TAR RNA determines the recruitment of human P-TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts (15,18,30,33,44,65).Binding of the 7SK small nuclear RNA (snRNA) to P-TEFb has recently been shown to be associated with the inhibition of CDK9 kinase activity...
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