P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

The entry of RNA polymerase II into a productive mode of elongation is controlled, in part, by the postinitiation activity of positive transcription elongation factor b (P-TEFb) (Marshall, N. F., and Price, D. H. (1995) J. Biol. Chem. 270, 12335-12338). We report here that removal of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II abolishes productive elongation. Correspondingly, we found that P-TEFb can phosphorylate the CTD of pure RNA polymerase II. Furthermore, P-TEFb can phosphorylate the CTD of RNA polymerase II when the polymerase is in an early elongation complex. Both the function and kinase activity of P-TEFb are blocked by the drugs 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and H-8. P-TEFb is distinct from transcription factor IIH (TFIIH) because the two factors have no subunits in common, P-TEFb is more sensitive to DRB than is TFIIH, and most importantly, TFIIH cannot substitute functionally for P-TEFb. We propose that phosphorylation of the CTD by P-TEFb controls the transition from abortive into productive elongation mode.

Production of full-length runoff transcripts in vitro and functional mRNA in vivo is sensitive to the drug 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). We previously proposed the existence of an activity, P-TEF (positive transcription elongation factor) that functions in a DRB-sensitive manner to allow RNA polymerase II elongation complexes to efficiently synthesize long transcripts (Marshall, N. F. and Price, D. H. (1992) Mol. Cell. Biol. 12, 2078-2090). We have fractionated nuclear extracts of Drosophila melanogaster Kc cells and identified three activities, P-TEFa, factor 2, and P-TEFb, that are directly involved in reconstructing DRB-sensitive transcription. P-TEFb is essential for the production of DRB-sensitive long transcripts in vitro, while P-TEFa and factor 2 are stimulatory. P-TEFb activity is associated with a protein comprising two polypeptide subunits with apparent molecular masses of 124 and 43 kDa. Using a P-TEFb-dependent transcription system, we show that P-TEFb acts after initiation and is the limiting factor in the production of long run-off transcripts.

We have examined elongation by RNA polymerase II initiated at a promoter and have identified two classes of elongation complexes. Following initiation at a promoter, all polymerase molecules enter an abortive mode of elongation. Abortive elongation is characterized by the rapid generation of short transcripts due to pausing of the polymerase followed by termination of transcription. Termination of the early elongation complexes can be suppressed by the addition of 250 mM KCI or 1 mg of heparin per ml soon after initiation. Elongation complexes of the second class carry out productive elongation in which long transcripts can be synthesized. Productive elongation complexes are derived from early paused elongation complexes by the action of a factor which we call P-TEF (positive transcription elongation factor). P-TEF is inhibited by 5,6-dichloro-1-B-Dribofuranosylbenzimidazole at concentrations which have no effect on the initiation of transcription. By using templates immobilized on paramagnetic particles, we show that isolated preinitiation complexes lack P-TEF and give rise to transcription complexes which can carry out only abortive elongation. The ability to carry out productive elongation can be restored to isolated transcription complexes by the addition of P-TEF after initiation. A model is presented which describes the role of elongation factors in the formation and maintenance of elongation complexes. The model is consistent with the available in vivo data concerning control of elongation and is used to predict the outcome of other potential in vitro and in vivo experiments.It is now clear that the transcription of eucaryotic genes is controlled during the elongation phase as well as at initiation. The number of genes for which elongational control has been implicated is growing (80). The three proto-oncogenes c-myc (52, 61, 65, 79, 85), c-myb (4, 62), and c-fos (16, 70) have been shown to be controlled at elongation. Adenovirus (37,66,73), simian virus 40 (36, 67), minute virus of mice (39), and human immunodeficiency virus (HIV) (40,41,75,83) have been demonstrated to have specific blocks to transcription elongation. The mRNA levels for the adenosine deaminase genes of humans and mice are at least partly controlled by a regulated block to elongation (13,14,42,48,58). Many genes in Drosophila melanogaster have RNA polymerase II molecules arrested early after initiation (68,69). While control of elongation has been implicated in these examples, very little is known about the molecular mechanisms involved.A number of factors that influence elongation and termination by procaryotic RNA polymerase have been defined (86). In particular, the N and Q protein-mediated antitermination systems of lambda and similar bacteriophages provide a model for how specific gene expression can be controlled by modifying RNA polymerase elongation. The mechanism by which lambda Q protein functions has been partially elucidated (87). A key feature in this process is the pausing of the RNA polymerase downstream of the initiation...

Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts. The purified factor can exist in two forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic Elp3 subunit. Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly with RNA polymerase II in solution. Several human homologues of the yeast Elongator subunits were identified as subunits of the human Elongator complex, including StIP1 (STAT-interacting protein 1) and IKAP (IKK complex-associated protein). Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this disease might be due to compromised Elongator function and therefore could be a transcription disorder.