Xin Lin, Ran Taube, Koh Fujinaga, and B. Matija Peterlin¶

From the Departments of Medicine, Microbiology, and Immunology, University of California at San Francisco, California 94143-0703

¶ To whom correspondence should be addressed: Rm. N215 UCSF-Mt. Zion Cancer Center, 2340 Sutter St., San Francisco, CA 94115.    Tel.: 415-502-1905;    Fax: 415-502-1901;
E-mail: matija@itsa.ucsf.edu

Different positive transcription elongation factor b (P-TEFb) complexes isolated from mammalian cells contain a common catalytic subunit (Cdk9) and the unique regulatory cyclins CycT1, CycT2a, CycT2b, or CycK. The role of CycK as a transcriptional cyclin was demonstrated in this study. First, CycK activated transcription when tethered heterologously to RNA, which required the kinase activity of Cdk9. Although this P-TEFb could phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAPII) in vitro, in contrast to CycT1 and CycT2, CycK did not activate transcription when tethered to DNA. Interestingly, when the C termini of CycT1 and CycT2 or only the histidine-rich stretch from positions 481 to 551 in CycT1 were added to CycK, the extended chimeras activated transcription equivalently via DNA. Moreover, these transcriptional effects required the CTD of RNAPII in cells. Thus, CycK functions as P-TEFb only via RNA, which suggests the presence of cellular RNA-bound activators that require CycK for their transcriptional activity.

The elongation step is critical for transcription by RNA polymerase II (RNAPII) (1). Many cellular factors have been identified for their roles in this process. They include P-TEFb and the negative transcription elongation factor (N-TEF) (1-3). In this scheme, RNAPII can initiate but not elongate because of its interaction with N-TEF
(2, 4). Most likely, N-TEF is composed of the 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF). DSIF contains SPT4 (14 kDa) and SPT5 (160 kDa) (5, 6). NELF contains four subunits, of which the NELF-E/RD subunit contains an RNA recognition motif (RRM) (7). P-TEFb is then recruited to the transcription complex, where its kinase subunit phosphorylates the C-terminal domain (CTD) of RNAPII (8, 9) and N-TEF (10, 11), allowing the elongation of
transcription to proceed. P-TEFb was first identified as the factor that was required for the reconstitution of DRB sensitivity in Drosophila melanogaster (8). Over the past 2 years, different P-TEFb complexes have been identified. These heterodimers contain a catalytic subunit, Cdk9, and a regulatory subunit, which can be CycT1, CycT2, or CycK (12-14). CycT2 exists in two forms, CycT2a and CycT2b, because of alternative splicing. CycT1, CycT2a, CycT2b, and CycK share extensive sequence similarity in their cyclin boxes at the N terminus from positions 1 to 250, which contain the Cdk9 binding domains. CycT1, CycT2a, and CycT2b also contain long C-terminal extensions, but their sequences diverge significantly. However, CycK is relatively small. It contains a short C-terminal domain of 107 residues from positions 251 to 357.

Recent findings revealed that P-TEFb, which contains CycT1 and Cdk9, is the key cellular factor that supports Tat transactivation and HIV replication (2, 15, 16). The human but not the murine P-TEFb supports the effects of Tat (12, 17-20). Tat is expressed early in the replicative cycle of HIV and is essential for viral gene expression and replication (21). It recognizes the 5'-bulge in the transactivation response (TAR) stem loop RNA, which is located at the 5'-end of all viral transcripts. Tat binds CycT1, and together, they form the combinatorial surface that interacts with the TAR RNA with high affinity and specificity (12). The obligate partner of CycT1, Cdk9, then phosphorylates the CTD of RNAPII. Thus, Tat promotes HIV transcription at the step of elongation rather than initiation (22). The key step in these effects of Tat is the recruitment of P-TEFb to RNAPII. These findings also raised the possibility that such a system may exist in higher eukaryotes where Tat homologs may recruit P-TEFb to RNA and activate transcription of cellular genes.

CycK was first identified as a protein that could restore progression through the cell cycle and was most closely related to human cyclins C and H (23). Reports have also suggested that CycK associates with a potent Cdk kinase activity (CAK) in vitro. Recently, the kinase partner of CycK was identified as Cdk9 (14). This complex between CycK and Cdk9 could also function as a CTD kinase in vitro (14). However, the role of CycK in transcriptional regulation has not been defined in vivo, and the question of whether CycK is solely a CAK or is also a transcriptional cyclin has not been answered. Additionally, this complex appears to play no role in Tat transactivation. Nevertheless, the understanding of other P-TEFb complexes should give us a more complete picture of the function of these transcription elongation factors.

In this study, the ability of CycK to activate transcription when recruited to a complete promoter via RNA or DNA was examined. First, we tethered CycK to RNA. Later, we fused the C termini of CycT1 or CycT2 (more precisely the histidine-rich stretch from CycT1 to the C terminus of CycK) and then tethered these hybrid proteins to DNA. We discovered that CycK can function as a transcriptional cyclin only via RNA, and the histidine-rich stretch from CycT1 confers its ability to function via DNA in vivo.
...

DISCUSSION

In this study, we have demonstrated that CycK forms an active P-TEFb complex with Cdk9 and promotes transcription via RNA in vivo. In sharp contrast, CycK could not activate transcription via DNA although it still functioned as a CTD kinase in vitro (14). Because CycT1 and CycT2 can activate transcription via DNA and the
histidine-rich stretch in CycT1 binds the CTD, the transcriptional activity of the hybrid Gal·CycK protein could be rescued by extending it with the C termini from CycT1 or CycT2. More importantly, the histidine-rich stretch from CycT1 alone could also rescue the activity of the Gal·CycK fusion protein via DNA. These extended tripartite fusion proteins required the CTD of RNAPII for activity. Thus, the C termini of these cyclin subunits from P-TEFb play important regulatory roles and dictate their substrate specificities.

It is to be noted that our studies were performed in vivo rather than in vitro. In in vitro transcription systems, P-TEFb is already present in the preinitiation complex, and the complex between CycK and Cdk9 functions via DNA (14). This finding could be due to very different stoichiometries of DNA templates and transcription complexes or compositions of nuclear extracts. Additionally, exogenously added, abundant P-TEFb could bind the CTD or RNAPII without the help of other proteins. Nevertheless, all other studies point to the obligatory recruitment of P-TEFb by RNA- or DNA-bound activators or their coactivators in cells. Thus, Tat, CIITA, NF-B, androgen receptor, and c-Myc all recruit P-TEFb to the transcription complex (12, 31-34). Only then does P-TEFb interact with the CTD of RNAPII and N-TEF, leading to their phosphorylation (8-11). This modification results in the transition from initiation to elongation of eukaryotic transcription. Importantly, CycK lacks this CTD-interacting domain. Because nascent RNA moves from the catalytic pocket in RNAPII along the CTD (35), Cdk9 bound to CycK can still phosphorylate this substrate. DNA presentation is qualitatively different, where P-TEFb must first find and bind the CTD for Cdk9 to phosphorylate the RNAPII. The histidine-rich stretches in CycT1 and CycT2 perform this function. This finding explains why all three cyclins function via RNA, but only CycT1 and CycT2 can activate transcription via DNA.

The theme of cyclins directing the activity of their associate kinases is not new. For example, the specificity of Cdk2 is governed by its associated cyclin A, especially by its hydrophobic MRAIL sequence, which is required for the binding and recognition of target proteins that contain the RXL motif (36). Likewise, CycK, CycT1, and CycT2 contain their highest sequence similarity in their cyclin boxes, where they bind Cdk9. Their C termini are very divergent. CycT1 and CycT2 share little besides the histidine-rich stretch, and only CycT1 possesses the TRM and PEST sequences (12-13). Additionally, only the complex between CycT1 and Cdk9 can activate HIV-1 transcription (12). The TRM sequence is required for this function (12). A recent study demonstrated the PEST sequence in CycT1 is required for the interaction between CycT1 and SCF (SKP2), which then targets Cdk9 for ubiquitination and degradation by the proteasome (37). Moreover this P-TEFb complex only phosphorylates CTD at serine 2 during HIV-1 transcription (38). In this study, the complex between CycK and Cdk9 could phosphorylate both serines 2 and 5. Thus, this P-TEFb might have a broader specificity. Supporting this notion,
CycK was first associated with a CAK activity (23). Other differences in the modes of action and target specificities of these different P-TEFb complexes will be revealed in future studies with many distinct activators and by following its genetic inactivation in the mouse.

Our studies with CycK also suggest the tantalizing possibility that other cellular activators, i.e. Tat homologs, exist that function via RNA. They are expected to bind CycK and recruit this P-TEFb complex to positions downstream from the site of initiation of transcription. Indeed, a strategy using the yeast three-hybrid screening system to detect RNA-protein interactions identified 70 RNA sequences that functioned independently of an exogenous activation domain on their associated proteins (39). Moreover, these RNA sequences needed to be positioned near the promoter for their effects (39). It is likely that these RNA species could fold into structures that directly recruited RNA-bound activators (39). Finally, CycK could also participate in cotranscriptional processing to maintain the hyperphosphorylated state of RNAPII before polyadenylation and maturation of primary transcripts. Such a cotranscriptional role for P-TEFb is also hinted at by recent studies, which suggest
that some complexes between CycT1 and Cdk9 associate with 7SK RNA (40, 41), which has been colocalized with U1 snRNA in the nucleus (42).

ACKNOWLEDGEMENTS

We thank Paula Zupanc-Ecimovic for secretarial assistance, Dan Irwin for technical help, Dirk Eick for reagents, and members of the Peterlin laboratory for helpful discussions and comments.

FOOTNOTES

* This work was supported by Grant RO1 AI49104-01 from the National Institutes of Health and Grant R00-SF-006 from the University-wide AIDS Research Program (UARP) (to M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a fellowship from the Campbell Foundation.

Supported by a fellowship from the UARP.

Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M200117200

ABBREVIATIONS

The abbreviations used are: RNAPII, RNA polymerase II; CTD, carboxyl terminal domain; TAR, transactivation response; HIV, human immunodeficiency virus; SL, stem loop; Rev, regulator of expression of virion genes; RRE, Rev response element; HA, hemagglutinin; GST, glutathione S-transferase; wt, wild-type; CAT, chloramphenicol acetyltransferase; TEF, transcription elongation factor; N-TEF, negative-TEF; P-TEF, positive-TEF; DRB, 5,6-dichloro-1-D-ribofuranosylbenzimidazole.

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