Control of formation of two distinct classes of RNA polymerase II elongation complexes.

Abstract

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...

RNA Polymerase II Elongation Control

“…As demonstrated by the examples in Figure 2A, in the production of mRNA a major restriction point occurs shortly after Pol II initiates. The polymerase becomes stably engaged by elongating the nascent transcript past about 12 nt and then comes under the control of factors that significantly slow or halt elongation (16). These promoter proximately paused polymerases are the major form of polymerase found bound to metazoan chromosomes and are poised for entry into productive elongation (9,17).…”

“…In vitro transcription experiments initially provided the tools to identify both the positive and negative factors involved in controlling the elongation phase of transcription (16). A key feature of transcription of virtually any promoter in vitro using a crude nuclear extract is the inhibition of the generation of long transcripts by the ATP analog 5,6-Dichloro-1-β-Dribofuranosylbenzimidazole (DRB) (16,22). P-TEFb (23), NELF (19) and DSIF (18) were purified based on their activities during reconstitution of DRB-sensitive transcription using fractionated nuclear extracts.…”

Regulation of eukaryotic gene expression by transcriptional attenuation.

“…The drug 5,6-dichloro-lb-D-furanosylbenzimidazole (DRB) was shown to inhibit production of transcripts elongating past the site of premature transcriptional termination in the c-myc gene in microinjected Xenopus oocytes; DRB had no effect on production of prematurely terminated transcripts, thus revealing two classes of transcription complex . Similarly, by analyzing transcriptional elongation on cloned templates in Drosophila cell extracts, Marshall and Price (1992) have identified two classes of elongation complex. Productive elongation complexes generating full length transcripts were shown to derive from early paused complexes by the action of a factor called P-TEF, which is inhibited by DRB (Kephart et al, 1992;Marshall and Price, 1992).…”

“…Similarly, by analyzing transcriptional elongation on cloned templates in Drosophila cell extracts, Marshall and Price (1992) have identified two classes of elongation complex. Productive elongation complexes generating full length transcripts were shown to derive from early paused complexes by the action of a factor called P-TEF, which is inhibited by DRB (Kephart et al, 1992;Marshall and Price, 1992). It was suggested that regulatory proteins could influence transcriptional elongation by enhancing the activity of P-TEF or a similar cellular factor.…”

Proapoptotic compound ARC targets Akt and N-myc in neuroblastoma cells

“…The nucleoside analog DRB (5,6-dichloro-1-b-Dribofuranosylbenzimidazole) is another transcriptional inhibitor, which like ARC, targets the elongation stage of transcription by inhibiting the positive transcription elongation factor b (P-TEFb, a complex of Cdk9/cyclin T1) (Marshall and Price, 1992). Previously, we had demonstrated that though ARC and DRB repressed transcription to a similar extent, ARC induced more potent apoptosis in a colon cancer cell line (Radhakrishnan and Gartel, 2006b).…”