The Sensitivity of RNA Polymerase II in Elongation Complexes to C-terminal Domain Phosphatase
2000; Elsevier BV; Volume: 275; Issue: 20 Linguagem: Inglês
10.1074/jbc.275.20.14923
ISSN1083-351X
AutoresAlan L. Lehman, Michael Dahmus,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe phosphorylation state of the carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of transcript elongation. This report examines the sensitivity of RNAP II to dephosphorylation by CTD phosphatase (CTDP) and addresses factors that regulate its sensitivity. The CTDP sensitivity of RNAP IIO in paused elongation complexes on a dC-tailed template does not significantly differ from that of free RNAP IIO. RNAP IIO contained in elongation complexes that initiate transcription from the adenovirus-2 major late promoter in the presence of a nuclear extract is relatively resistant to dephosphorylation. Complexes treated with 1% Sarkosyl remain elongation-competent but demonstrate a 5-fold increase in CTDP sensitivity. Furthermore, the sensitivity of RNAP IIO in both control and Sarkosyl-treated elongation complexes is dependent on their position relative to the start site of transcription. Elongation complexes 11–24 nucleotides downstream are more sensitive to dephosphorylation than complexes 50–150 nucleotides downstream. The incubation of Sarkosyl-treated elongation complexes with nuclear extract restores the original resistance to dephosphorylation. These results suggest that a conformational change occurs in RNAP II as it clears the promoter, which results in an increased resistance to dephosphorylation. Furthermore, the sensitivity to dephosphorylation can be modulated by a factor(s) present in the nuclear extract. The phosphorylation state of the carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of transcript elongation. This report examines the sensitivity of RNAP II to dephosphorylation by CTD phosphatase (CTDP) and addresses factors that regulate its sensitivity. The CTDP sensitivity of RNAP IIO in paused elongation complexes on a dC-tailed template does not significantly differ from that of free RNAP IIO. RNAP IIO contained in elongation complexes that initiate transcription from the adenovirus-2 major late promoter in the presence of a nuclear extract is relatively resistant to dephosphorylation. Complexes treated with 1% Sarkosyl remain elongation-competent but demonstrate a 5-fold increase in CTDP sensitivity. Furthermore, the sensitivity of RNAP IIO in both control and Sarkosyl-treated elongation complexes is dependent on their position relative to the start site of transcription. Elongation complexes 11–24 nucleotides downstream are more sensitive to dephosphorylation than complexes 50–150 nucleotides downstream. The incubation of Sarkosyl-treated elongation complexes with nuclear extract restores the original resistance to dephosphorylation. These results suggest that a conformational change occurs in RNAP II as it clears the promoter, which results in an increased resistance to dephosphorylation. Furthermore, the sensitivity to dephosphorylation can be modulated by a factor(s) present in the nuclear extract. RNA polymerase adenovirus-2 major late promoter carboxyl-terminal domain CTD phosphatase 5,6-dichloro-1-β-d-ribofuranosylbenzimidaze DRB sensitivity-inducing factor negative elongation factor positive transcription elongation factor b the 74-kDa subunit of TFIIF polyacrylamide gel electrophoresis bovine serum albumin milliunits RNA polymerase (RNAP)1II is a large multisubunit enzyme responsible for catalyzing the transcription of protein coding genes in eukaryotes. The largest subunit of RNAP II contains at its C terminus a unique and highly conserved domain composed of tandem repeats of the consensus sequence YSPTSPS (for a review, see Ref. 1.Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Genetic analysis of the C-terminal domain (CTD) has established that it is essential for viability (for a review, see Ref. 2.Carlson M. Annu. Rev. Cell. Dev. Biol. 1997; 13: 1-23Crossref PubMed Scopus (179) Google Scholar), although it is dispensable for transcription from some promoters in vitro. Two forms of the enzyme existin vivo that differ with respect to the phosphorylation of the CTD. RNAP IIA is unphosphorylated, whereas RNAP IIO is highly phosphorylated (3.Cadena D.L. Dahmus M.E. J. Biol. Chem. 1987; 262: 12468-12474Abstract Full Text PDF PubMed Google Scholar). RNAP IIA assembles into preinitiation complexes with the general transcription factors (4.Reinberg D. Orphanides G. Ebright R. Akoulitchev S. Carcamo J. Cho H. Cortes P. Drapkin R. Flores O. Ha I. Inostroza J.A. Kim S. Kim T.K. Kumar P. Lagrange T. LeRoy G. Lu H. Ma D.M. Maldonado E. Merino A. Mermelstein F. Olave I. Sheldon M. Shiekhattar R. Zawel L. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 83-103Crossref PubMed Scopus (55) Google Scholar, 5.Chesnut J.D. Stephens J.H. Dahmus M.E. J. Biol. Chem. 1992; 267: 10500-10506Abstract Full Text PDF PubMed Google Scholar, 6.Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 1993; 62: 161-190Crossref PubMed Scopus (344) Google Scholar, 7.Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Crossref PubMed Scopus (390) Google Scholar). Although multiple protein kinases can phosphorylate the CTD (for a review, see Ref. 1.Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar), the initial phosphorylation is catalyzed by a protein kinase intrinsic to the preinitiation complex (8.Coin F. Egly J.M. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 105-110Crossref PubMed Scopus (51) Google Scholar, 9.Lu H. Zawel L. Fisher L. Egly J.M. Reinberg D. Nature. 1992; 358: 641-645Crossref PubMed Scopus (330) Google Scholar). The idea that the phosphorylation of the CTD results in a disruption of the protein-protein interactions that initially brought RNAP IIA to the preinitiation complex remains an attractive but unproven hypothesis. Transcript elongation is catalyzed by RNAP IIO. Completion of the transcription cycle is dependent on the dephosphorylation of RNAP IIO, a reaction that may be coupled to transcript termination. CTD phosphatase (CTDP) has been characterized in yeast and mammalian cells (10.Archambault J. Chambers R.S. Kobor M.S. Ho Y. Cartier M. Bolotin D. Andrews B. Kane C.M. Greenblatt J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14300-14305Crossref PubMed Scopus (128) Google Scholar, 11.Archambault J. Pan G. Dahmus G.K. Cartier M. Marshall N. Zhang S. Dahmus M.E. Greenblatt J. J. Biol. Chem. 1998; 273: 27593-27601Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 12.Chambers R.S. Dahmus M.E. J. Biol. 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Reinberg D. Genes Dev. 1999; 13: 1540-1552Crossref PubMed Scopus (172) Google Scholar) demonstrates that, when transcription is initiated in a defined system, the ternary elongation complex can be dephosphorylated by CTD phosphatase. Increasing evidence suggests that an interplay of positive and negative factors regulate transcript elongation. Although the role of the CTD in elongation remains unclear, the CTD appears to be the regulatory focus of many protein factors. Distinct structural changes occur in early elongating RNAP II between +25 and +40 (17.Luse D.S. Samkurashvili I. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 289-300Crossref PubMed Scopus (9) Google Scholar, 18.Samkurashvili I. Luse D.S. Mol. Cell. Biol. 1998; 18: 5343-5354Crossref PubMed Scopus (44) Google Scholar). Furthermore, in this same region, the elongation factor P-TEFb can phosphorylate the CTD, resulting in a stabilization of the elongation complex (19.Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). This reaction is inhibited by the nucleotide analogue DRB. The failure of P-TEFb to act can lead to abortive transcription. In addition to P-TEFb, several negative factors coordinately regulate the transition from abortive to productive elongation. DSIF was initially characterized as a protein factor required to reconstitute DRB sensitivity in vitro (20.Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (566) Google Scholar). In the absence of DRB, DSIF represses transcription and antagonizes the positive action of P-TEFb (21.Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). The negative effect of DSIF depends on the state of CTD phosphorylation. Prior phosphorylation of the CTD by TFIIH or P-TEFb should result in transcription complexes that are resistant to the effects of DSIF (20.Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (566) Google Scholar, 22.Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (280) Google Scholar). Interestingly, both NELF and DSIF are required to repress transcript elongation, although neither functions to repress transcription by RNAP IIO. This indicates that if RNAP becomes dephosphorylated during the course of transcription, DSIF and NELF can repress transcription (21.Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). Recent results suggest that CTDP can play a direct role in the regulation of transcript elongation. The human immunodeficiency virus type 1 transcriptional activator, Tat, interacts with and inhibits the activity of CTD phosphatase (23.Marshall N.F. Dahmus G.K. Dahmus M.E. J. Biol. Chem. 1998; 273: 31726-31730Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Conversely, P-TEFb is recruited by Tat (24.Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N.F. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (612) Google Scholar, 25.Herrmann C.H. Rice A.P. J. Virol. 1995; 69: 1612-1620Crossref PubMed Google Scholar, 26.Herrmann C.H. Gold M.O. Rice A.P. Nucleic Acids Res. 1996; 24: 501-508Crossref PubMed Scopus (58) Google Scholar, 27.Zhou Q. Sharp P.A. Science. 1996; 274: 605-610Crossref PubMed Scopus (139) Google Scholar). Accordingly, the presence of Tat leads to a high level of CTD phosphorylation, resulting in a highly processive RNAP II and the efficient expression of the viral genome. These results indicate that the elongation efficiency of RNAP II is regulated at least in part by protein kinases and phosphatase(s) that establish the level of CTD phosphorylation. The objective of these studies is to gain insights into the factors that regulate the sensitivity of RNAP IIO in elongation complexes to dephosphorylation. The results presented suggest that RNAP IIO in early elongation complexes is more sensitive to dephosphorylation than is RNAP IIO in elongation complexes that have cleared the promoter. Resistance to dephosphorylation appears to involve both a conformational change in RNAP II and the association of a specific factor(s). Buffer A contained 50 mm Tris-HCl, pH 7.9, and 0.1 mm EDTA. Buffer B contained 50 mm Tris-HCl, pH 7.9, 0.1 mm EDTA, 5 mm MgCl2, 0.5 mm dithiothreitol, 20 mm KCl, 0.025% Tween 80, and 20% glycerol. Buffer C contained 50 mm Tris-HCl, pH 7.9, 0.1 mm EDTA, 5 mm MgCl2 0.5 mm dithiothreitol, 20% glycerol, and KCl as indicated. Buffer D contained 25 mm Tris, pH 7.9, 5 mmMgCl2, 0.5 mm dithiothreitol, 0.025% Tween 80, 20% glycerol, and KCl as indicated. RNAP IIA was purified from calf thymus by the method of Hodo and Blatti (28.Hodo H.G. Blatti S.P. Biochemistry. 1977; 16: 2334-2343Crossref PubMed Scopus (130) Google Scholar) with the modifications described by Kang and Dahmus (29.Kang M.E. Dahmus M.E. J. Biol. Chem. 1993; 268: 25033-25040Abstract Full Text PDF PubMed Google Scholar). RNAP IIA was labeled with 32P as described in Chamberset al. (13.Chambers R.S. Wang B.Q. Burton Z.F. Dahmus M.E. J. Biol. Chem. 1995; 270: 14962-14969Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Casein kinase II was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). 32P-Labeled RNAP IIO for dC-tailed transcription and control CTDP reactions was preparedin vitro by the phosphorylation of 32P-labeled RNAP IIA as described by Marshall and Dahmus (23.Marshall N.F. Dahmus G.K. Dahmus M.E. J. Biol. Chem. 1998; 273: 31726-31730Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To produce the DNA templates, 0.5 μg of pUC HTXB (30.Dahmus M.E. Kedinger C. J. Biol. Chem. 1983; 258: 2303-2307Abstract Full Text PDF PubMed Google Scholar) was subjected to 30 rounds of polymerase chain reaction with 2 μm concentrations of the following primers: forward 5′-TTCCCAGTCACGACGTTGTA-3′ and reverse 5′-CACAGGAAACACGTATGACC-3′. The reaction buffer was 20 mm Tris, pH 7.9, 50 mmKCl, 1.5 mm MgCl2, 200 μm dATP, dCTP, dGTP, and dTTP in a 2-ml reaction divided into 100-μl aliquots. This results in a template DNA 945 base pairs in length that will give a promoter-dependent run-off transcript of 622 nucleotides. The polymerase chain reaction was loaded onto a 1-ml MonoQ column (Amersham Pharmacia Biotech) and eluted with a 20-ml linear gradient of 0.1–1 m NaCl in buffer A. The HTXB DNA fragment elutes at ∼0.75 m NaCl. Promoter-independent transcription on dC-tailed templates was performed using templates produced with a biotinylated reverse primer. Promoter-dependent transcription was carried out on templates biotinylated on the forward primer. Promoter-independent DNA templates (dC-tailed) were generated from 5 μg of HTXB. The template was cut with SacI (Life Technologies, Inc.) following the manufacturer's instructions, after which the DNA was phenol/chloroform- and chloroform-extracted, and ethanol-precipitated. After resuspension in buffer A, a tailing reaction was assembled with 38 units of terminal deoxynucleotide transferase (Promega) and 1 mm dCTP (Amersham Pharmacia Biotech) following the manufacturer's instructions in a reaction volume of 100 μl. After 15 min of incubation (sufficient to add 60–90 dCMPs to the 3′-OH generated by SacI), the tailed DNA reaction was adjusted to 750 mm NaCl and bound to Dynabeads (Dynal) as described by the manufacturer. 4.2 μg of DNA was added per 100 μl of Dynabeads previously equilibrated in buffer A. The reaction was incubated at 22 °C for 60 min in buffer A containing 750 mm NaCl while turning on a rotator. Dynabeads were washed free of unbound DNA with buffer A and resuspended at a DNA concentration of 20 ng/μl in buffer A. About 95% of input DNA was bound to the beads. Transcription extracts were purified as described by Laybourn and Dahmus (31.Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1990; 265: 13165-13173Abstract Full Text PDF PubMed Google Scholar) with the following changes. HiTrap heparin (Amersham Pharmacia Biotech) was substituted for heparin-Sepharose, and the primary transcription extract (DE0.25) and RNA polymerase II (DE0.6) containing fractions were eluted from the DEAE-5PW (Waters) with 0.25 and 0.6 m KCl, respectively. TFIIA does not bind to the heparin column (HS0.24 fraction) and was added back separately from the other general transcription factors found in the DE0.25 fraction. Standard dC-tailed transcription reactions were initiated by the addition of 0.25 pmol of32P-labeled RNA polymerase IIO in ∼2 μl of buffer D containing ∼500 mm KCl to 100 ng of dC-tailed HTXB DNA immobilized on Dynabeads (as described above) in the presence of 0.5 μg/μl BSA in buffer C containing no MgCl2. Final reaction conditions prior to the addition of nucleotides were equivalent to buffer C containing 56 mm KCl, 0.5 μg/μl BSA, and no MgCl2 in a 20-μl reaction volume. After incubation at 30 °C for 30 min, ATP, UTP, and GTP were added to 0.6 mm, and CTP and MgCl2 were added to 10 μm and 6 mm, respectively. The final reaction volume was 25 μl. Five μCi of [α-32P]CTP (Amersham Pharmacia Biotech) was included in reactions carried out to determine the size distribution of RNA transcripts and to quantify their amount. RNAP IIO was allowed to elongate for 10 min and then washed free of nucleotides with buffer C containing 50 mm KCl. If digestion of nascent RNA was required, 1 unit of RNase H (Life Technologies, Inc.) was added, and reactions were incubated for 10 min at 37 °C. RNA products or RNAP II was visualized as described below. Preinitiation complexes were formed in a standard reaction by the addition of 1 μl of HiTrap heparin flow-through in buffer C with 100 mm KCl (contains TFIIA), 5 μl of DE0.25 in buffer C with 50 mm KCl (contains remaining general transcription factors), 0.25 pmol of32P-RNAP IIA in ∼1 μl of buffer D containing ∼500 mm KCl, and 100 ng of template DNA conjugated with Dynabeads. The reaction was adjusted to 56 mm KCl in a final volume of 20 μl in buffer C prior to incubation for 30 min at 30 °C. After incubation, 0.6 mm ATP and UTP, 250 nm CTP, 6 mm MgCl2, and 50 mm KCl were added for a total reaction volume of 25 μl. Five μCi of [α-32P]CTP was added when radiolabeled RNA was to be analyzed. Complete reactions were incubated for 2 min at 30 °C. Transcription was stopped as specified in the figure legends either by the addition of 15 mm EDTA or by twice washing the transcription complexes to remove free nucleotides. Beginning with washed pulse complexes in a 20-μl volume, chase elongation complexes were formed by the addition of ATP, UTP, and GTP to 0.6 mm and CTP to 10 μm in buffer C with 200 mm KCl and 0.5 mg/ml BSA in a final volume of 25 μl. Complexes were allowed to elongate for 1 min at 30 °C before being washed in buffer C with 50 mm KCl. Radiolabeled RNAP II in transcription complexes was visualized by SDS-PAGE carried out according to the method of Laemmli (32.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207123) Google Scholar) with a 5% polyacrylamide resolving gel. Radiolabeled RNA transcripts were analyzed by urea-PAGE as described by Chesnut et al. (5.Chesnut J.D. Stephens J.H. Dahmus M.E. J. Biol. Chem. 1992; 267: 10500-10506Abstract Full Text PDF PubMed Google Scholar) utilizing 12.5% polyacrylamide. Specific RNA transcripts were quantitated on a Fuji phosphor imager by comparing radioactive signal intensity of transcript bands to known amounts of [α-32P]CTP standard. The sensitivity of RNAP IIO in elongation complexes was determined by a modification of the assay described by Chamberset al. (13.Chambers R.S. Wang B.Q. Burton Z.F. Dahmus M.E. J. Biol. Chem. 1995; 270: 14962-14969Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Unless otherwise specified, RAP74 was included in all CTDP assays. Input radiolabeled RNAP IIO in transcription complexes on immobilized DNA was washed twice in buffer B. CTDP was purified as described previously (23.Marshall N.F. Dahmus G.K. Dahmus M.E. J. Biol. Chem. 1998; 273: 31726-31730Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Assays were quantitated on a Fuji phosphor imager in the following fashion. The region of the gel ranging from just above the subunit IIo band to just below the subunit IIa band was subdivided into approximate thirds. These thirds were designated IIo, int, and IIa for the regions corresponding to the subunit IIo and the intermediate and subunit IIa bands and were quantitated each as a percentage of the whole after subtraction of a lane-specific background (sample taken from beneath the subunit IIa band). Ten fmol of RNAP (∼1 μl) was removed from the reaction mix and suspended in Laemmli buffer just prior to the first wash to serve as a quantitation reference standard. The reference standard is removed prior to the first wash to account for the ∼15% nonspecific loss of CKII label during the preincubation of the 32P-labeled RNAP with transcription extract. To establish a base line for the sensitivity of RNAP IIO, purified 32P-labeled RNAP IIO was incubated with increasing concentrations of CTD phosphatase (Fig.1 A, lanes 1–5). The amount of subunits IIo and IIa were quantitated from phosphor imager scans and plotted against mU of CTD phosphatase (Fig. 1 B, left panel). The 50% conversion of RNAP IIO to IIA occurs at 9.5 mU of CTD phosphatase. Transcription was then initiated on a dC-tailed template immobilized on paramagnetic beads, and the CTD phosphatase sensitivity of RNAP IIO in elongation complexes was established. Analysis of RNA transcripts indicates that at 10 min RNAP IIO is distributed from about 150 to 650 nucleotides downstream from the site of initiation (Fig.1 C). The CTD phosphatase sensitivity of RNAP IIO in these elongation complexes does not differ appreciably from that of free RNAP IIO (Fig. 1 A, compare lanes 1–5 withlanes 11–15, and in Fig. 1 B, compareleft and right panels). Accordingly, the formation of an elongation complex per se does not result in protection of the CTD from dephosphorylation. Furthermore, the addition of free DNA and paramagnetic beads does not appreciably alter the sensitivity of RNAP IIO (Fig. 1, A andB, lanes 6–10 and center panel, respectively). Lanes 1–10contained 0.06 pmol of RNAP IIO, whereas lanes 11–15 contained about 0.13 pmol of RNAP IIO in elongation complex. The amount of CTDP required for 50% dephosphorylation of free RNAP IIO, free RNAP IIO in the presence of template DNA and Dynabeads, and RNAP IIO in transcription complexes is 9.5, 17, and 23 milliunits, respectively (Table I).Table IThe CTDP sensitivity of RNAP IIOSource of RNAP IIORNAP II50% conversionfmolmU CTDPFig. 1 RNAP IIO609.5 RNAP IIO + dC-tailed templateadC-tailed template bound to Dynabeads.6017 Elongation complex on dC-tailed template13023Fig. 2 Elongation complex4.41000Fig. 4 Control elongation complexbNumbers are the average of two independent experiments.Pulse6.1660Chase1.81100 1% Sarkosyl-treated elongation complexbNumbers are the average of two independent experiments.Pulse0.5126Chase0.39200Fig. 6 Pulse Sarkosyl-treated elongation complex0.4612 Pulse Sarkosyl-treated elongation complex − RAP740.53440The left-hand column denotes the source of the RNAP IIO. All CTDP reactions were carried out in the presence of RAP74 except where indicated (− RAP74). The middle column presents the amount of RNAP IIO either free or in elongation complex calculated from the reference standards. Data in the middle column are calculated from the 0-mU CTDP reaction. The column on the right displays the calculated mU of CTDP required to dephosphorylate 50% of the input RNAP IIO. The relative amount of subunit IIo at 0 mU CTDP was taken as 100%.a dC-tailed template bound to Dynabeads.b Numbers are the average of two independent experiments. Open table in a new tab The left-hand column denotes the source of the RNAP IIO. All CTDP reactions were carried out in the presence of RAP74 except where indicated (− RAP74). The middle column presents the amount of RNAP IIO either free or in elongation complex calculated from the reference standards. Data in the middle column are calculated from the 0-mU CTDP reaction. The column on the right displays the calculated mU of CTDP required to dephosphorylate 50% of the input RNAP IIO. The relative amount of subunit IIo at 0 mU CTDP was taken as 100%. Promoter-independent transcription on dC-tailed templates produces transcripts in a RNA-DNA hybrid, which prevents the DNA strands from reannealing behind RNA polymerase (33.Kadesch T.R. Chamberlin M.J. J. Biol. Chem. 1982; 257: 5286-5295Abstract Full Text PDF PubMed Google Scholar). To account for differences in the sensitivity of dC-tailed complexes that might arise from these RNA-DNA hybrids, RNase H was employed to digest the hybridized RNA, thereby allowing the reannealing of the DNA template. Digestion of the nascent RNA does not alter the CTDP sensitivity of RNAP IIO (data not shown). Promoter-dependent transcription was carried out utilizing a DNA fragment containing the Ad2-MLP immobilized on Dynabeads. Preinitiation complexes were formed with32P-labeled RNAP IIA in the presence of a partially fractionated nuclear extract free of endogenous RNAP II. ATP, CTP, and UTP were added, and the reaction was incubated for 2 min to allow the production of short transcripts. Although the first G is at position 11, the predominant product is 11 nucleotides in length and corresponds to a pause due to limited CTP (see Fig. 3 B, lane 1, for an equivalent reaction). Following incubation, 10 fmol of RNAP II was removed from the reaction (Fig.2 A, lane 0). The bead-bound complexes were then washed in phosphatase buffer to remove both unbound extract and unbound RNAP II. The RNAP II bound to immobilized DNA is shown in lane 1. Less than 1% of the input RNAP II is recovered in complex. Furthermore, although the major fraction of RNAP II in the reaction is IIA (lane 0), the major fraction of RNAP II associated with the DNA has been converted to RNAP IIO (lane 1). Quantitation of the amount of transcript produced relative to the amount of RNAP II bound indicates that 12–50% of RNAP II bound under these conditions produces a transcript.Figure 2CTD phosphatase sensitivity of early elongation complexes initiated from the Ad2-MLP. To provide a reference point for the sensitivity of RNAP IIO in elongation complexes, transcription was initiated in the presence of a nuclear extract on the Ad2-MLP. Early elongation complexes, prepared in the absence of GTP, were purified and assayed for sensitivity to CTDP.A, lane 0 is a 10-fmol RNAP II reference standard. Lane 1 is a reaction stopped prior to incubation with CTDP. Lanes 2 and 3 are the bound and wash fractions, respectively, resulting from a 30-min control incubation in buffer B. Lanes 4–9 are identical to lanes 2 and3 except for the presence of increasing amounts of CTDP as indicated and 35 pmol of RAP74. Lanes 10 and 11are identical to lanes 8 and 9 except for the inclusion of exogenous radiolabeled RNAP IIO as a control for CTDP activity. Lanes 12 and 13 are purified reference RNAP IIO incubated in the absence and presence of CTDP as indicated.B, quantitation of the amount of subunits IIo and IIa, as well as the intermediate region (int) as described under “Experimental Procedures.” Numbers above eachgraph correspond to the lane in A from which each set of numbers was obtained.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The washed RNAP II complexes were incubated with increasing amounts of CTDP, in the presence of 35 pmol of RAP74. At the end of the CTDP reaction, the complexes were washed, resulting in two populations of RNAP. Both populations were resolved by SDS-PAGE, and the sensitivity of the bound population was determined by quantitation of subunits IIo and IIa (Fig. 2 B). The bound fraction (Fig. 2 A,even lanes 2–10) remains attached to the template DNA while the wash fraction (odd lanes 3–11) is released. Only RNAP IIA is found in the wash fraction even in the absence of CTDP (Fig. 2 A,lane 3). Although some dephosphorylation is seen, complete dephosphorylation is not obtained with the highest concentrations of CTDP tested under these conditions (Fig.2 A, lane 8). As a control, the reaction containing 400 mU of CTDP was run in duplicate in the absence (lanes 8 and 9) and presence (lanes 10 and 11) of exogenous32P-RNAP IIO. The finding that free RNAP IIO is not protected from dephosphorylation, as indicated by the absence of a subunit IIo band in the wash fraction (lane 11), indicates that protection is not conferred by a transacting factor. These results suggest that RNAP II in complexes formed in the presence of a nuclear extract is about 50-fold more resistant to dephosphorylation than complexes formed on tailed templates (Table I). However, the interpretation of these results is complicated by the fact that the substrate for dephosphorylation is a mixed population of RNAP IIO, only some of which is in functional elongation complexes. A characterization of the CTD phosphatase sensitivity of RNAP IIO in elongation complexes is dependent on the analysis of a homogenous population of functional complexes. This is assured only if the molar amount of transcript produced equals the molar amount of RNAP II in complex. In an effort to remove nonproductively bound RNAP II, complexes were treated with Sarkosyl. Transcription was initiated on immobilized DNA in the presence of an RNAP II-depleted nuclear extract supplemented with 32P-labeled RNAP IIA. Furthermore, in reactions to determine the size distribution of RNA and to quantify the amount of transcript produced,
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