P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo

Abstract

Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.RNA polymerase II (Pol II) undergoes a series of critical modifications as it progresses through a cycle of transcription. The carboxyl-terminal domain (CTD) of the largest subunit of Pol II, which consists of tandem heptapeptide repeats with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , is a major target for these modifications, which include phosphorylation (primarily of serines 2 and 5 of the consensus repeat), proline cis/trans isomerization, and glycosylation (36). Distinct phosphorylation marks are associated with different transcription stages: in metazoans, Pol II enters the preinitiation complex in a hypophosphorylated state; it progresses from the initiation site to promoter-proximal regions, where it is phosphorylated primarily at Ser5; it matures into a doubly (Ser2 and Ser5) phosphorylated, productively elongating Pol II; and upon passing the polyadenylation site, it displays reduced phosphorylation (7,20,30). These phosphorylation states allow Pol II to interact with distinct repertoires of proteins at different stages of the transcription cycle and are critical in the coordination of transcription and coupled pre-mRNA processing (44,46). The Cdk7 and Cdk9 kinases are responsible for most CTD serine phosphorylation in metazoans (39). Cdk7, a subunit of the general transcription factor TFIIH, phosphorylates the CTD preferentially at Ser5 (47,53,60). By contrast, Cdk9, which together with cyclin T composes the P-TEFb kinase complex (42), phosphorylates the CTD predominantly at Ser2 (39, 47, 57, 71). P-TEFb and Cdk7 both have positive roles in transcription and function at postinitiation steps (3,22,33,56). P-TEFb recruitment to an activated gene is concomitant with the efficient release of Pol II from a promoter-proximally paused to a product...

Functional integration of transcriptional and RNA processing machineries

“…This step coincides with the initial RNAPII pausing event ∼30 nt downstream the transcriptional start. The initial pausing is also thought to be a checkpoint for the recruitment of CTD Ser2 kinases, such as P-TEFb, for RNAPII to enter the productive mode of transcriptional elongation [5,21]. In contrast to the capping enzymes, a large of number of RNA proteins (Table 1) implicated in RNA splicing and 3′ end formation bind to CTD in a Ser2 or Ser5/Ser2 phosphorylation dependent manner [22][23][24][25][26][27].…”

Roles of enhancer of zeste homolog 2: From skeletal muscle differentiation to rhabdomyosarcoma carcinogenesis

“…Initially, the transactivator Six4 recruits UTX to muscle-specific genes promoters, resulting in the demethylation of regulatory regions upstream the transcription start site; afterwards, removal of the repressive H3K27me3 mark within the coding region needs elongating RNA Polymerase II (RNA Pol II), established by pTEFIIb-dependent RNA Pol II phosphorylation. 135,136 Furthermore, recent studies demonstrated that the demethylation process is dependent on Spt6, a histone chaperone that destabilizes histone dimmer-tetramer nucleosomal contacts promoting transcriptional activation. Spt6 orchestrates H3K27 demethylation interacting with UTX and RNA Pol II at chromatin regions of muscle-specific genes, and it is required for appropriate muscle gene expression and myogenesis.…”

The transition from transcriptional initiation to elongation

“…It is presumed that HSF recruits factors that release paused Pol II and permit it to traverse the gene, although it is unclear which factors are direct targets and exactly how Pol II release occurs. p-TEFb plays an important role, because artificial recruitment bypasses the pause [57], and chemical inhibition blocks release from the pause [58]. It is likely that other activators (e.g.…”