Topological Localization of the Carboxyl-Terminal Domain of RNA Polymerase II in the Initiation Complex

Douziech, Maxime; Forget, Diane; Greenblatt, Jack; Coulombe, Benoit

doi:10.1074/jbc.274.28.19868

Cited by 14 publications

(10 citation statements)

References 49 publications

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“…Binding to TAR RNA could serve to position the P-TEFb complex adjacent to the RNA exit channel, which is implicated from structural studies to lie near the RNAPII CTD (10). We show here that Tat can promote phosphorylation of the CTD by CycT1-CDK9 through binding to the phosphorylated substrate via the arginine-rich motif and bridging of the P-TEFb complex to its substrate.…”

Section: Discussionmentioning

confidence: 82%

CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

Garber

Mayall

Suess

et al. 2000

Molecular and Cellular Biology

148

171

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, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.Activation of human immunodeficiency virus type-1 (HIV-1) transcription by the virus-encoded transcription factor, Tat, provides an important paradigm for understanding the mechanisms that regulate transcription elongation by RNA polymerase II (RNAPII). Transcription complexes that form at the HIV-1 promoter in the absence of Tat are competent to initiate transcription but elongate inefficiently, due to the effects of negative general elongation factors (22,50,51,55,57; reviewed in references 16 and 56) and an inhibitory RNA structure that induces pausing of RNAPII complexes (38). Tat functions as a promoter-specific transcription elongation factor through binding to the transactivation response element (TAR) in the 5Ј-untranslated leader of viral transcripts to stimulate processive transcription by RNAPII (for a review, see references 29 and 30).Tat regulates an early step in transcription elongation that requires cyclin T1 (CycT1) and CDK9 (21,35,40,52,(58)(59)(60), which are subunits of the positive transcription elongation factor P-TEFb (36) and Tat-associated kinase (21, 23, 24) complexes. CDK9 is a Cdc2-related kinase (20) that promotes general elongation of transcription at many promoters in vitro and can phosphorylate the C-terminal domain (CTD) of the largest subunit of RNAPII (9,35,60). We previously cloned CycT1 as a protein that interacts strongly with the 48-amino-

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Section: Discussionmentioning

confidence: 82%

CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

Garber

Mayall

Suess

et al. 2000

Molecular and Cellular Biology

148

171

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“…6). Because the CTD and the RNA exit channel are closely juxtaposed in the initiation complex (39,40), transit through the CTD may facilitate their unloading to the nascent pre-mRNA. If not loaded rapidly after transcription, secondary structure formation on the pre-mRNA may compromise the recognition of splicing signals by splicing factors.…”

Section: Discussionmentioning

confidence: 99%

A Human RNA Polymerase II-containing Complex Associated with Factors Necessary for Spliceosome Assembly

Robert¹,

Blanchette²,

Maës³

et al. 2002

Journal of Biological Chemistry

Self Cite

111

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Transcription and splicing are coordinated processes in mammalian cells. We have used affinity chromatography with immobilized transcription elongation factor SII to purify a protein complex that contains core RNA polymerase II (RNA Pol II), the general transcription initiation factors, and several splicing factors, including the U1, U2, and U4 small nuclear RNPs, the U2AF 65 , and serine/arginine-rich proteins. The splicing factors and the transcription machinery co-purify through a gel filtration column and co-immunoprecipitate in experiments using an anti-U2AF 65 antibody, indicating that they are part of a unique complex. Although the RNA Pol II-containing complex does not possess splicing activity, it can complement small nuclear RNP-inactivated extracts and can promote the formation of a pre-spliceosome complex. Because interactions between components of the splicing and transcription machineries occur in the context of a complex containing a hypophosphorylated RNA Pol II capable of initiating transcription, our results suggest that the coupling between transcription and splicing begins before transcription initiation.The splicing of pre-mRNA in mammalian cells is the nuclear process during which introns are removed from primary transcripts synthesized by RNA polymerase II (RNA Pol II).1 Splicing is achieved by the spliceosome, a large macromolecular complex, composed of small nuclear ribonucleoprotein (snRNP) particles and additional proteins including members of the serine/arginine-rich (SR) protein family (reviewed in Refs. 1 and 2). Cytological studies have revealed that splicing can occur in a co-transcriptional manner (see Refs. 3 and 4 for examples) and that factors necessary for splicing are recruited to sites of transcription (5-7). A growing body of data indicates that coupling between transcription and a variety of RNA maturation events may be achieved through the C-terminal domain (CTD) of the RPB1 subunit of RNA Pol II (reviewed in Refs. 8 -11). The CTD is a seven-amino acid motif tandemly repeated 52 times in humans and 26 -27 times in yeast. It is highly conserved among eukaryotic organisms and is subject to reversible phosphorylation during the transcription cycle (reviewed in Refs. 12 and 13). Two isoforms of RNA Pol II exist in vivo, namely RNA Pol IIA and IIO. RNA Pol IIA possesses a hypophosphorylated CTD and preferentially enters the preinitiation complex, whereas RNA Pol IIO harbors an extensively phosphorylated CTD and is found in the elongation complex (13,14). Biochemical studies indicate that RNA Pol II physically interacts with components involved in capping (15-18), polyadenylation (19), and splicing (20 -24). Whereas a phosphorylated CTD is essential for the interaction with capping enzymes, the polyadenylation factor CPSF is brought to the transcription complex through an interaction with TFIID and transferred to the phosphorylated CTD after transcription initiation (19,25). The phosphorylated RNA Pol IIO isoform has been found to associate with splicing factors and has been i...

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“…In preinitiation complexes the CTD is buried close to the DNA template and surrounded by basal transcription factors, including TFIIH, and proteins of the mediator complex. Cross-linking studies have shown that in preinitiation complexes the polymerase is orientated with CTD making contacts with the DNA template at around position 16, downstream of the transcription start site (15). The repeat unit of each heptapeptide in the CTD contains two SPXX motifs (SPTS and SPSY) that allow the CTD to fold into a compact beta-turn stabilized by a side chain-main chain interaction (5).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

Kim

Bourgeois

Isel

et al. 2002

Molecular and Cellular Biology

153

134

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Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-␤-D-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-␤-D-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.Transcription in eukaryotic cells is regulated by a series of phosphorylation events involving the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) (for a review, see reference 55). Initially, Pol II with a hypophosphorylated CTD (Pol II a ) is recruited to the promoter. The hypophosphorylated CTD is able to form a tightly packed complex at the promoter, together with general initiation factors and the mediator complex. During initiation, the CTD becomes highly phosphorylated, creating a form of the enzyme called Pol II o . The large conformational change induced in the enzyme during the transition from initiation to early elongation releases the phosphorylated CTD and permits it to interact with a variety of elongation factors, as well as with proteins involved in mRNA processing (28).The CTD comprises 52 tandem repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Three cyclindependent kinases-CDK7, CDK8, and CDK9-are known to phosphorylate the CTD during transcription (55). The three CTD kinases primarily pho...

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Topological Localization of the Carboxyl-Terminal Domain of RNA Polymerase II in the Initiation Complex

Cited by 14 publications

References 49 publications

CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

A Human RNA Polymerase II-containing Complex Associated with Factors Necessary for Spliceosome Assembly

Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

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