As polymerase elongates further downstream, levels of Ser5P drop and Ser2P levels increase. It is important to note that lower levels of Ser5P persist throughout elongation, so at least some CTD repeats in transcribing RNApII are likely to be phosphorylated at both Ser2 and Ser5 (and possibly also Ser7). The drop in Ser5P is not dependent upon Ser2 phosphorylation, since Ser5P does not increase in cells in which Ser2 phosphorylation is blocked (Cho et al., 2001
; Liu et al., 2009
; Qiu et al., 2009
; Zhou et al., 2009
). Several recent discoveries help clarify the events mediating the CTD transition as RNApII moves into later elongation.
Mosley et al. (2009)
recently identified the long-postulated Ser5P phosphatase, an evolutionarily conserved RNApII-binding protein called Rtr1. Deletion of Rtr1 leads to persistently high Ser5P levels during elongation. Interestingly, Rtr1 is not essential for viability in yeast, although this may be due to the presence of a related and genetically redundant protein (Rtr2). Ssu72 is an essential transcription termination factor that also has Ser5P phosphatase activity (Krishnamurthy et al., 2004
), but Ssu72 mutants have not been found to affect the promoter-proximal Ser5P drop. One possibility is that Rtr1 reduces Ser5P in early elongation phase, while Ssu72 removes the remaining Ser5P further downstream during termination. It is also worth noting that another Ser5P phosphatase called Scp1 has been identified in metazoans as a repressor of gene expression in neurons (Zhang et al., 2006
). Perhaps this enzyme dephosphorylates the CTD at a very early step before initiation or capping can occur.
Although the correlation between Ser2P and elongation was discovered a number of years ago (Komarnitsky et al., 2000
), recent discoveries have uncovered interesting complexities in the responsible kinases. In mammalian cells, the Cdk9 kinase subunit of the positive elongation factor P-TEFb phosphorylates CTD Ser2 as well as the elongation factor Spt4/Spt5 (also known as DSIF) (Peterlin and Price, 2006
). Reminiscent of the RNApII CTD, Spt5 contains multiple repeats of a short sequence containing the phosphorylation site. However, the Spt5 repeat differs from that of the RNApII CTD, so it will be interesting to see how the same kinase active site recognizes both substrates.
Yeast have two kinases that resemble mammalian Cdk9. It has been proposed that Cdk9 function is split in S. cerevisiae, with Ctk1 responsible for CTD Ser2 phosphorylation (Cho et al., 2001
) and Bur1 phosphorylating Spt4/5 (Zhou et al., 2009
). However, recent papers indicate the story is not so simple (Liu et al., 2009
; Qiu et al., 2009
). These new studies show that, although Ctk1 provides the bulk of Ser2 phosphorylation, Bur1 also contributes to Ser2P just downstream of the promoter and this early phosphorylation helps promote more extensive phosphorylation by the second kinase. Therefore, much like mammalian Cdk9, Bur1 phosphorylates both the CTD and Spt5 to contribute to effective transcription. Similarly, the S. pombe Bur1 homolog (SpCdk9) can phosphorylate both CTD and Spt5, while the Ctk1 homolog (Lsk1) contributes the majority of Ser2 phosphorylation (Viladevall et al., 2009
). Given that Bur1/spCdk9 resembles metazoan Cdk9 in targeting both Spt5 and CTD Ser2 during early elongation, it's interesting to consider whether higher eukaryotes may have a second kinase that functions like Ctk1/Lsk1 to more extensively phosphorylate Ser2 further downstream.
What triggers the progression of kinases during the transition from initiation to elongation? Several plausible mechanisms have been described by which Ser5P would help recruit the Ser2 kinases. In S. cerevisiae, Bur1 directly binds to the Ser5P CTD, providing a very simple mechanism for having Ser2P come after Ser5P (Qiu et al., 2009
). In S. pombe, Cdk9 is complexed with the mRNA cap methyltransferase. Since capping enzymes bind the Ser5P CTD, and methylation is the final step of capping, this coupling makes Ser2 phosphorylation dependent upon earlier events (Viladevall et al., 2009
). In turn, Ctk1 helps release RNApII from the basal initiation factors, including TFIIH (Ahn et al., 2009
). Given that CTD Ser2 and Spt5 phosphorylation are critical for later events (see below), these interactions may constitute a “5′ checkpoint” that makes the activity of the Ser2/Spt5 kinases contingent upon Ser5P and mRNA capping ().
Levels of CTD Ser2P increase gradually as RNApII moves away from the promoter. Several mechanisms appear to contribute to this gradient. First, there appears to be ongoing dephosphorylation of Ser2P during elongation by the Fcp1 phosphatase, particularly in 5′ regions where the basal factor TFIIF may stimulate Fcp1 activity (Cho et al., 2001
). Second, the kinetics of CTD phosphorylation are not uniform and may be slow compared to transcript elongation. In vitro experiments suggest that early phosphorylation events can “prime” the CTD for more efficient subsequent modification. Both SpCdk9 (Viladevall et al., 2009
) and S. cerevisiae Ctk1 (Jones et al., 2004
) are much more active on CTD substrates that already have some phosphorylation. Several possible mechanisms for stimulation can be envisioned. There could be a non-catalytic phosphoserine binding site on the kinase that tethers the enzyme to the substrate for efficient CTD phosphorylation (i.e. processivity). An even simpler model is that the early phosphates cause the CTD to adopt an extended conformation that makes it a more accessible substrate. In either case, Ser2P phosphorylation may accelerate while the polymerase is elongating, causing Ser2P to peak further downstream.
Phosphorylation at CTD Ser2 appears to mark RNApII molecules that are competent for the long-range elongation necessary to produce most mRNAs. Indeed, at many non-expressed genes, RNApII is seen only at the promoter and carries Ser5P but not Ser2P (reviewed in Margaritis and Holstege, 2008
). How does the CTD transition affect elongation? Phosphorylation of the CTD does not directly affect elongation rate, but instead mediates interactions between the polymerase and other factors. Therefore, the Ser5P to Ser2P transition could either promote the association and activity of positive elongation factors, or inhibit pathways that cause RNApII to pause or terminate early in elongation. In fact, both types of mechanisms appear to operate.