The RNAP II CTD heptapeptide Tyr1
contains three serine residues at positions 2, 5, and 7. The CTD is heavily phosphorylated in vivo, and substitution of nonphosphorylatable amino acids at position 2 or 5 of the Saccharomyces cerevisiae
CTD is lethal (77
). Phosphorylation of the CTD is temporally linked to the transition between transcription initiation and elongation. Human CTD kinases specific for serine 5, serines 2 and 5, or serines 2 and 7 have been characterized (11
). It has been reported that the TFIIH kinase phosphorylates serine 5 of the CTD (58
). The analyses of TFIIH kinase activity in HIV-1 transcription complexes presented in this manuscript are consistent with these findings. The substrate specificity of the P-TEFb complex has been more difficult to distinguish. Ramanathan et al. have recently reported that serine 5 is essential for phosphorylation by the Tat-TAK complex (Tat–P-TEFb complex) (55
). Using an in vitro kinase assay, the investigators demonstrated that phosphorylation of the CTD peptide was abolished when a mutation was introduced at serine 5. Mutation of serines 2 and 7 did not affect the activity of the Tat-TAK complex. The authors did not include reactions which contained P-TEFb alone in the absence of Tat. Thus, it was not obvious that the serine 5 phosphorylation observed in their assay is the Tat-modified function of P-TEFb. Moreover, the specificity of the P-TEFb kinase alone cannot be distinguished. Patturajan et al. have recently reported that the deletion of yeast CTDK-I, a CDK kinase closely related to P-TEFb, eliminates the increase in CTD serine 2 phosphorylation during a response to nutrient depletion (48
). In our studies, we provide clear evidence that recombinant P-TEFb phosphorylates the CTD at serine 2. In the presence of Tat, the substrate specificity of P-TEFb is altered such that it phosphorylates serine 2 and serine 5. It is important to point out that the change in substrate specificity is reproduced in the natural setting of the kinase, the transcription complex, and the natural substrate, the RNAP II CTD.
A unique feature of Tat transactivation of HIV-1 transcription has been observed, which is that it is preferentially inhibited by DRB, an adenosine analogue that targets RNAP II-mediated elongation. P-TEFb has been distinguished from other transcription factors by its sensitivity to very low doses of DRB. In this regard, the results presented in this report demonstrate that serine 2 phosphorylation and serine 5 phosphorylation by Tat-activated P-TEFb are sensitive to DRB and that at a concentration of 5 μM DRB, serine 2 and serine 5 phosphorylation was inhibited by approximately 70 or 90%, respectively.
It will be of interest to determine whether the initial phosphorylation at serine 2 and serine 5 within the PICs is important for Tat transactivation. Okamoto et al. (44
) have shown that the CTD is not required for basal transcription and for the formation of short, attenuated transcripts. In contrast, transcriptional activation by Tat in vivo and in vitro requires the CTD. It is possible that the critical step of CTD phosphorylation takes place in the elongation complex, after CDK7 has been released, TAR RNA has been synthesized, and Tat–P-TEFb has been recruited to the complex. TAR likely acts to recruit the Tat–P-TEFb complex to the elongation complex in this activation process. It will also be of interest to determine whether phosphorylation of serine 2, serine 5, or serines 2 and 5 is required for Tat transactivation at this stage.
In a very elegant analysis of the fate of transcription factors during the transition from initiation to elongation, Zawel et al. have demonstrated that TFIID remains promoter bound, wherease TFIIB, TFIIE, TFIIF, and TFIIH are released rapidly (83
). TFIIH release occurs after the complex reaches +30 to +50. Consistent with these studies, our analysis indicates that TFIIH is released between +14 and +36 (Fig. A). Previous observations suggest that Tat interacts with a target cellular protein (as a cofactor of Tat) and that the interaction of Tat with its cellular cofactor is a prerequisite for TAR binding (1
). Recent studies strongly imply that the Tat cofactor is a cellular protein kinase termed TAK which interacts with the activation domain of Tat and phosphorylates CTD (21
). It has been found that a human positive-acting transcription elongation factor complex called P-TEFb is actually equivalent to TAK (85
). Several observations further suggest that the recruitment of the Tat–P-TEFb complex by TAR binding to elongating complexes is the critical step in Tat transactivation (22
). Similar to the results obtained in this study, recent observations indicate that the entry of the Tat–P-TEFb complex into the transcription complex comes at the time of PIC assembly (47
). Our results presented here are consistent with the observations that Tat–P-TEFb associates with HIV-1 PICs. Importantly, in contrast to the TFIIH-CDK7 complex, which is released from the complex between +14 and +36, P-TEFb remains stably associated with the TECs. The results presented in this study further suggest that the ability of Tat to alter the substrate specificity of CDK9 would allow the continued hyperphosphorylation of the RNAP II CTD at serine 2 and serine 5 in the Tat transcription elongation complex. As a general transcription elongation factor, it will also be important to determine how P-TEFb acts on other promoters and whether other activators affect the activity of P-TEFb.
Genetic data from several groups show that TAR can be functionally replaced by heterologous RNA structures. The subsequent recruitment of Tat to these RNA targets by fusion of Tat to an RNA binding domain can clearly fully activate HIV-1 LTR-dependent transcription (59
). The results suggest that TAR acts only as an interface. Further, the recruitment of P-TEFb to an HIV-1 LTR containing a heterologous promoter-proximal target by fusion of cyclin T1 to an RNA binding domain is both necessary and sufficient for full activation of transcription from HIV-1 LTR in vivo. The authors of the latter study suggest that Tat does not activate the P-TEFb in any specific way but rather serves as an interface between the RNA and the enzyme complex (3
). The results presented in the latter study demonstrate that Tat, in fact, does modify the activity of the P-TEFb-associated CDK9 kinase, altering the specificity of CTD phosphorylation to allow CDK9 to phosphorylate serine 5.
Multiple kinases appear to be involved in phosphorylating the CTD in vivo (7
). In yeast, at least three distinct complexes have been described, Kin28-CCL1 (8
), SRB10-SRB11 (36
), and CTDK1 (a possible yeast homologue of P-TEFb) (48
). In higher eukaryotes, there are three homologues, CDK7-cyclin H (14
), CDK8-cyclin C (34
), and P-TEFb (40
). The observations demonstrate that CDK8-cyclin C (SRB10-SRB11, the yeast homologue) associates with the RNAP II holoenzyme (34
). However, only a small portion (less than 10% in mammals and 6% in yeast) of total RNAP II was found to be associated in a holoenzyme form with CDK8 (SRB10) in vivo (30
). It has been reported that SRB10-SRB11 (CDK8-cyclin C) is a negative regulator of transcription (20
) and that SRB10 is not a general repressor of protein-coding genes (36
). CTD phosphorylation by SRB10-SRB11 kinase prevents the assembly of the PIC and thereby represses the transcription of specific genes (20
), including those involved in cell type specificity (74
), meiosis (64
), sugar utilization (31
), and stress response (9
). Our results presented in this report suggest that CDK8 may not function in HIV-1 transcription and Tat transactivation.
Finally, it is of interest to consider the possibility that phosphorylation of the RNAP II CTD may have a direct effect on capping of the pre-mRNAs. Capping is targeted to nascent RNAs through binding of the guanyltransferase to the phosphorylated CTD. Guanyltransferase binds CTD peptides containing phosphate groups at either serine 2 or serine 5. Interestingly, it has recently been reported that binding of guanyltransferase to CTDs containing a phosphorylated serine 5 specifically stimulates enzymatic activity by enhancing the affinity for GTP and increasing the yield of enzyme-GMP intermediate (23
). A CTD containing phosphorylated serine 2 has no effect on enzymatic activity. It will be of importance to determine if the Tat-directed P-TEFb phosphorylation at serine 5 contributes to the capping of the viral pre-mRNA. Several studies have previously shown that Tat enhances the translation of mRNAs synthesized from the HIV-1 LTR (10
). As capping is known to markedly increase the efficiency of translation of mRNAs, it will be interesting to determine whether Tat enhances recruitment of capping enzymes to the hyperphosphorylated CTD.