A Highly Purified RNA Polymerase II Elongation Control System
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m104967200
ISSN1083-351X
AutoresDan B. Renner, Yuki Yamaguchi, Tadashi Wada, Hiroshi Handa, David H. Price,
Tópico(s)Virus-based gene therapy research
ResumoStudying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole has led to the discovery of a number of proteins involved in the regulation of transcription elongation by RNA polymerase II. We have developed a highly purified elongation control system composed of three purified proteins added back to isolated RNA polymerase II elongation complexes. Two of the proteins, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), act as negative transcription elongation factors by increasing the time the polymerase spent at pause sites. P-TEFb reverses the negative effect of DSIF and NELF through a mechanism dependent on its kinase activity. TFIIF is a general initiation factor that positively affects elongation by decreasing pausing. We show that TFIIF functionally competes with DSIF and NELF, and this competition is dependent on the relative concentrations of TFIIF and NELF. Studying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole has led to the discovery of a number of proteins involved in the regulation of transcription elongation by RNA polymerase II. We have developed a highly purified elongation control system composed of three purified proteins added back to isolated RNA polymerase II elongation complexes. Two of the proteins, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), act as negative transcription elongation factors by increasing the time the polymerase spent at pause sites. P-TEFb reverses the negative effect of DSIF and NELF through a mechanism dependent on its kinase activity. TFIIF is a general initiation factor that positively affects elongation by decreasing pausing. We show that TFIIF functionally competes with DSIF and NELF, and this competition is dependent on the relative concentrations of TFIIF and NELF. 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole human immunodeficiency virus HeLa nuclear extract 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor negative elongation factor dithiothreitol phenylmethylsulfonyl fluoride nucleotide The balance of activity between both positive and negative factors achieves accurate control of many cellular processes. Accumulating evidence indicates that such a process regulates the control of transcription elongation (1Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (571) Google Scholar). It has been proposed that shortly after initiation, negative transcription elongation factors act upon RNA polymerase II to cause production of short transcripts (2Marshall N.F. Price D.H. Mol. Cell. Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (241) Google Scholar). With the action of P-TEFb the polymerase enters productive elongation and transcription is no longer influenced by the negative factors (3Marshall N.F. Price D.H. J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 4Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). After this transition, the polymerase is acted upon by general elongation factors such as S-II, TFIIF, ELL, and elongin to generate long transcripts (5Aso T. Conaway J.W. Conaway R.C. FASEB J. 1995; 9: 1419-1428Crossref PubMed Scopus (56) Google Scholar, 6Shilatifard A. FASEB J. 1998; 12: 1437-1446Crossref PubMed Scopus (99) Google Scholar, 7Reines D. Conaway R.C. Conaway J.W. Curr. Opin. Cell Biol. 1999; 11: 342-346Crossref PubMed Scopus (69) Google Scholar). The key step in the elongation control process, the transition from abortive elongation to productive elongation, requires the positive elongation factor P-TEFb (1Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (571) Google Scholar, 3Marshall N.F. Price D.H. J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 4Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). P-TEFb was originally purified from Drosophila nuclear extracts, as a factor required for reconstitution of 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)1 sensitivity in in vitro transcription assays (3Marshall N.F. Price D.H. J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Active human P-TEFb consists of a heterodimer of cyclin-dependent kinase 9 (Cdk9) and either cyclin T1, cyclin T2, or cyclin K (8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (451) Google Scholar, 9Fu T.J. Peng J. Lee G. Price D.H. Flores O. J. Biol. Chem. 1999; 274: 34527-34530Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The elongation properties of P-TEFb are dependent on its kinase activity, and both the kinase and elongation activities are sensitive to the nucleotide analog DRB (3Marshall N.F. Price D.H. J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 4Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (451) Google Scholar, 10Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (613) Google Scholar, 11Mancebo H.S. Lee G. Flygare J. Tomassini J. Luu P. Zhu Y. Peng J. Blau C. Hazuda D. Price D.H. Flores O. Genes Dev. 1997; 11: 2633-2644Crossref PubMed Scopus (480) Google Scholar, 12Peng J. Marshall N.F. Price D.H. J. Biol. Chem. 1998; 273: 13855-13860Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), a kinase inhibitor known for its ability to inhibit transcription elongation (13Chodosh L.A. Fire A. Samuels M. Sharp P.A. J. Biol. Chem. 1989; 264: 2250-2257Abstract Full Text PDF PubMed Google Scholar). P-TEFb is also strongly inhibited by flavopiridol, a drug currently in clinical trials as an anti-cancer treatment that might also be useful as an anti-HIV therapy (14Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Depletion of P-TEFb from HeLa nuclear extract (HNE) greatly reduces the ability of RNA polymerase II to produce full-length transcripts and eliminates the DRB sensitivity of that extract (8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (451) Google Scholar). The addition of purified P-TEFb to HNE depleted of Cdk9 restores the ability of RNA polymerase II to generate full-length transcripts and restores DRB sensitivity (8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (451) Google Scholar, 15Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (281) Google Scholar). Another factor required for DRB sensitivity, DRB sensitivity-inducing factor (DSIF), was purified based on its ability to reconstitute DRB sensitivity in a partially purified transcription system (16Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (571) Google Scholar). DSIF is composed of the 14-kDa Spt4 subunit (p14) and the 120-kDa Spt5 subunit (p160) (17Yamaguchi Y. Narita T. Inukai N. Wada T. Handa H. J. Biochem. (Tokyo). 2001; 129: 185-191Crossref PubMed Scopus (43) Google Scholar). Spt4 possesses a putative zinc finger domain and probably interacts with Spt5 through mainly hydrophobic interactions (18Yamaguchi Y. Wada T. Watanabe D. Takagi T. Hasegawa J. Handa H. J. Biol. Chem. 1999; 274: 8085-8092Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Spt5 has a highly acidic N-terminal region, four copies of the KOW repeat, and two sets of repeats in the C-terminal region (CTR1 and CTR2). RNA polymerase II has been shown to directly interact with Spt5 through a region encompassing the KOW repeats (18Yamaguchi Y. Wada T. Watanabe D. Takagi T. Hasegawa J. Handa H. J. Biol. Chem. 1999; 274: 8085-8092Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 19Ivanov D. Kwak Y.T. Guo J. Gaynor R.B. Mol. Cell. Biol. 2000; 20: 2970-2983Crossref PubMed Scopus (177) Google Scholar). KOW repeats are also found in the Escherichia coli elongation control protein NusG (20Kyrpides N.C. Woese C.R. Ouzounis C.A. Trends Biochem. Sci. 1996; 21: 425-426Abstract Full Text PDF PubMed Scopus (112) Google Scholar). NusG binds the prokaryotic RNA polymerase and is required for certain specific termination and anti-termination activities (21Li J. Horwitz R. McCracken S. Greenblatt J. J. Biol. Chem. 1992; 267: 6012-6019Abstract Full Text PDF PubMed Google Scholar,22Sullivan S.L. Gottesman M.E. Cell. 1992; 68: 989-994Abstract Full Text PDF PubMed Scopus (150) Google Scholar). Saturating amounts of NusG also enhance the elongation rate of the E. coli RNA polymerase by ∼20% (23Burova E. Hung S.C. Sagitov V. Stitt B.L. Gottesman M.E. J. Bacteriol. 1995; 177: 1388-1392Crossref PubMed Google Scholar). Genetic and biochemical studies of the yeast, fruit fly, and zebrafish homologs of Spt5 have also linked these proteins to the regulation of transcription elongation (24Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (373) Google Scholar, 25Guo S. Yamaguchi Y. Schilbach S. Wada T. Lee J. Goddard A. French D. Handa H. Rosenthal A. Nature. 2000; 408: 366-369Crossref PubMed Scopus (141) Google Scholar, 26Zorio D.A. Bentley D.L. Curr. Biol. 2001; 11: R144-R146Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Depletion of DSIF from HNE greatly reduces the DRB sensitivity of that extract without significantly affecting the ability of RNA polymerase II to generate long transcripts (16Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (571) Google Scholar). Depletion of P-TEFb from a HNE eliminates DRB sensitivity and greatly reduces the ability of RNA polymerase II to generate long transcripts (8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (451) Google Scholar). Immunodepletion of both DSIF and P-TEFb from an extract restores the ability of RNA polymerase II to make long transcripts while eliminating the DRB sensitivity of that extract (15Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (281) Google Scholar). A dose-dependent add-back of purified DSIF and P-TEFb reconstitutes DRB-dependent elongation control (15Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (281) Google Scholar). This finding suggests that P-TEFb functions to reverse the negative activity associated with DRB sensitivity and that this negative activity is dependent on the presence of DSIF. The expression of a dominant negative form of DSIF in HeLa cells increased the expression level of each of four reporter genes severalfold (18Yamaguchi Y. Wada T. Watanabe D. Takagi T. Hasegawa J. Handa H. J. Biol. Chem. 1999; 274: 8085-8092Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), suggesting that DSIF is involved in general repression of transcription. The search for factors required for the reconstitution of DRB sensitivity in a partially purified transcription assay led to the discovery of a third factor, negative elongation factor (NELF) (27Yamaguchi 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 (622) Google Scholar). NELF consists of four subunits. The smallest of these, NELF-E, was cloned and found to contain repeats of the dipeptide Arg-Asp (RD repeats), an RNA recognition motif, and a leucine zipper at the C-terminal end (27Yamaguchi 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 (622) Google Scholar). Immunodepletion of NELF-E from an HNE significantly reduced the DRB sensitivity of that extract, while an add-back of purified NELF restored the DRB sensitivity to original levels. Using a deoxycytosine-tailed template assay, the effect of combining DSIF and NELF together with purified RNA polymerase II was tested (27Yamaguchi 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 (622) Google Scholar). Under these conditions, transcription was nearly completely inhibited. When DSIF and NELF were added to polymerases that had been paused on a tailed template, further elongation was also significantly reduced (27Yamaguchi 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 (622) Google Scholar). To further study the role of DSIF, NELF, and P-TEFb in elongation control, an assay using isolated RNA polymerase II elongation complexes was utilized. With this system, elongation control mediated by P-TEFb was reproduced in both a crude system, incorporating a HeLa nuclear extract and in a highly purified system, using only purified factors. We found that DSIF and NELF work cooperatively to decrease the elongation rate of the polymerase by increasing the time it spends at pause sites. The original elongation rate of the polymerase was restored by the addition of P-TEFb to the assay. We also show that NELF functionally competes with TFIIF in the presence of DSIF. Generation of pET constructs for the expression of both human Spt4 and Spt5 with an N-terminal His tag is described elsewhere (16Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (571) Google Scholar). Baculovirus expression and purification of P-TEFb (Cdk9 and cyclin T2a) are described by Peng et al. (8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (451) Google Scholar). Anti-FLAG M2-agarose affinity gel (A-1205) and FLAG peptide (F-3290) were obtained from Sigma. Recombinant human TFIIF was purified as described in Peng et al. (28Peng J. Liu M. Marion J. Zhu Y. Price D.H. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 365-370Crossref PubMed Scopus (31) Google Scholar). E. coli DE3 cells transformed with plasmids expressing Spt4 and Spt5 were grown at 37 °C in LB medium containing 100 μg/ml ampicillin. Because the expression of Spt5 had a negative effect on the growth of the cells, ampicillin selection was maintained by washing the cells in fresh medium several times during growth. When a liter of culture reached an absorbance of 0.5 absorbance units, it was induced with 400 μm isopropyl-1-thio-β-d-galactopyranoside. After a 3-h induction, cells were washed with PBS, resuspended in lysis buffer (PBS, 1% Triton X-100, 200 μm EDTA, 1 mm DTT, 0.1% of a saturated solution of PMSF in isopropyl alcohol), and sonicated. The lysate was spun at 50,000 × g for 40 min, the supernatant was removed, and the pellet was resuspended in urea buffer (25 mm HEPES, 100 μm EDTA, 100 mm KCl, 6 murea). The urea-extracted pellet was spun at 15,000 ×g for 30 min. 3.2 mm CaCl2 was added to complex the EDTA immediately before loading onto a 4-ml Ni2+-nitrilotriacetic acid-agarose column (Qiagen). The column was washed with 10 column volumes of wash buffer (10 mm Tris, 0.5 m NaCl, 1% Triton X-100, 6m urea, 5 mm imidazole) and eluted with wash buffer containing 200 mm imidazole. Purified Spt5 was combined with an approximately 10-fold excess of purified Spt4 and dialyzed into renaturation buffer (25 mm HEPES, 15% glycerol, 150 mm KCl, 100 μm EDTA, 0.1% PMSF saturated in isopropyl alcohol, and 1 mm DTT). Renatured Spt4 and Spt5 (DSIF) was loaded onto a Mono Q column equilibrated with 150 mm HGKEDP (HEPES (pH 7.6), 15% glycerol, 150 mm KCl, 0.1 mm EDTA, 1 mm DTT, and 0.1% of a saturated solution of PMSF in isopropyl alcohol). DSIF eluted at 400 mm KCl during a gradient from 150 to 800 mm HGKEDP. We found that the activity of purified DSIF decreased each time a sample was frozen and thawed due to precipitation. Because of this, all samples were frozen and thawed only once. To assist in the purification of the complete NELF protein, NELF-E was FLAG-tagged at the N terminus and inserted into the mammalian expression vector pCAGGS to generate pCMV-FLAG-NELF-E. Stably transfected HeLa S3 cells were selected and maintained in the presence of 500 μg/ml Geneticin. Large, mycoplasma-free cultures were grown in Joklik’s medium containing 5% calf serum by the National Cell Culture Center. 50 liters of cells were spun down, washed with PBS, and snap frozen for transportation. Cells were resuspended into isotonic lysis buffer (25 mm HEPES, 150 mm NaCl, 0.1% Nonidet P-40, 0.1% PMSF-saturated isopropyl alcohol, and 1 mm DTT) and homogenized using a Dounce homogenizer. The lysate was spun at 4000 × g for 10 min, and the cytoplasmic fraction was removed. The nuclear fraction was used to prepare a HeLa nuclear extract using the same protocol used earlier for the generation of Drosophila Kc cell nuclear extract (29Price D.H. Sluder A.E. Greenleaf A.L. J. Biol. Chem. 1987; 262: 3244-3255Abstract Full Text PDF PubMed Google Scholar). NELF was purified from both the cytoplasmic fraction and from 10% of the HNE as described below. The rest of the HNE was used for in vitrotranscriptions. The cytoplasmic fraction from 12 liters of the cells was brought to a NaCl concentration of 500 mm and loaded onto a 3-ml column of anti-FLAG M2-agarose affinity gel. The column was washed with 15 volumes of high salt buffer (25 mm HEPES, 500 mm NaCl, 0.1% Nonidet P-40, 0.1% PMSF-saturated isopropyl alcohol, and 1 mm DTT) and again with 5 volumes of low salt buffer (25 mm HEPES, 15% glycerol, 150 mmNaCl, 0.1% Nonidet P-40, 100 μm EDTA). NELF was eluted with 5 ml of low salt buffer containing 100 μg/ml FLAG peptide. Further purification of NELF was obtained by chromatography on a 1-ml Mono Q column. The column was equilibrated with 150 mmHGKEDP with 0.1% Nonidet P-40 and eluted with a 20-ml gradient to 1m HGKEDP with 0.1% Nonidet P-40. The NELF complex eluted at 360 mm. A DNA template containing the full cytomegalovirus promoter was generated by polymerase chain reaction of the pGL2CMV plasmid with a 5′-biotinylated primer to produce a 1387-base pair product. The DNA was incubated with streptavidin-labeled superparamagnetic beads (Dynabeads M-280) according to the manufacturer’s protocol to generate an immobilized template with a 548-nt runoff. For each experiment early elongation complexes were generated in one large reaction, isolated, and aliquoted into individual elongation reactions. For each final elongation reaction needed, 8 μl of preincubation mixture containing 200 ng of template and 1.6 μl of HNE was incubated with 20 mmHEPES, 60 mm KCl, 7 mm MgCl2, and 50 μm DRB for 10 min at room temperature (28Peng J. Liu M. Marion J. Zhu Y. Price D.H. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 365-370Crossref PubMed Scopus (31) Google Scholar). Transcription was initiated during a 30-s pulse with the addition of ATP, GTP, and UTP to 500 μm and 5 μCi of [α-32P]CTP for each final elongation reaction. Elongation was halted by the addition of EDTA to 25 mm. Complexes associated with the immobilized template were stringently washed three times with HKS (20 mm HEPES, 1 mKCl, and 1% Sarkosyl), two times with HKB (20 mm HEPES, 60 mm KCl, and 200 μg/ml bovine serum albumin), and resuspended in HKB. Extension of transcripts in early elongation complexes was carried out in 19-μl reactions by first mixing the isolated complexes with HNE or purified factors in the presence of 20 mm HEPES, 60 mm KCl, 200 μg/ml bovine serum albumin, and 1.1 units/μl RNasin (Promega). After 3–5 min, elongation was resumed by the simultaneous addition of NTPs to 500 μm and MgCl2 to 7 mm. Reactions were allowed to elongate for the indicated amounts of time at room temperature and were stopped by the addition of 200 μl of Sarkosyl Stop Solution (100 mm Tris, 100 mm NaCl, 10 mm EDTA, 1% Sarkosyl, 200 μg/ml tRNA). RNA preparation and analysis on 6% denaturing gel was described previously (29Price D.H. Sluder A.E. Greenleaf A.L. J. Biol. Chem. 1987; 262: 3244-3255Abstract Full Text PDF PubMed Google Scholar). To further our understanding of elongation control by P-TEFb, we developed an in vitro transcription system in which factors could be added back to isolated early elongation complexes. The attachment of the template to magnetic beads through a biotin-streptavidin linkage allows for the isolation of the early elongation complexes. The template is incubated with HNE to form preinitiation complexes on the CMV immediate early promoter. Upon the addition of nucleotides, including limiting [α-32P]CTP, RNA polymerase II initiates and generates transcripts predominately less than 25 nt in length. The elongating polymerases are halted and stripped of associated factors by repeatedly washing with buffer containing 1 m KCl and 1% Sarkosyl and resuspended in transcription buffer without nucleotides. The addition of nuclear extract to such complexes has been demonstrated to reconstitute P-TEFb-dependent elongation control (28Peng J. Liu M. Marion J. Zhu Y. Price D.H. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 365-370Crossref PubMed Scopus (31) Google Scholar). Here we use isolated elongation complexes to determine whether highly purified DSIF, NELF, and P-TEFb could reconstitute elongation control in a similar manner. We first examined the kinetics of RNA polymerase II elongation in the presence of a nuclear extract added back to early elongation complexes in the absence or presence of DRB as diagrammed in Fig. 1 A. Previous work, which is briefly described in the Introduction, strongly indicates that DRB sensitivity observed in reconstituted transcription assays is due exclusively to the inhibition of P-TEFb (1Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (571) Google Scholar, 4Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (451) Google Scholar, 12Peng J. Marshall N.F. Price D.H. J. Biol. Chem. 1998; 273: 13855-13860Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). After add-back of HNE, the early elongation complexes were chased for 0, 20, 30, 50, 80, 130, 210, or 340 s (Fig. 1 B). At the first few time points, little difference was observed in the elongation potential of the transcription complexes with or without DRB present. At longer time points, DRB reduced the elongation potential of a large fraction of the polymerases. This kinetic delay to the function of P-TEFb suggests that the kinase only functions on complexes that have either traveled a certain distance down the template or that P-TEFb requires a certain period of time to function. In the presence of DRB, complexes continued elongation, but at a much slower rate. The kinetics of elongation and the timing of P-TEFb function seen here match what was found previously using a system in which initiation and elongation were not separated by isolation of the early elongation complexes (2Marshall N.F. Price D.H. Mol. Cell. Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (241) Google Scholar). The results presented here demonstrate that the add-back assay faithfully reconstitutes appropriate kinetic aspects of elongation control. The two subunits of DSIF, Spt4 and Spt5, were both expressed in E. coli and purified as described under “Experimental Procedures” and summarized in Fig. 2 A. Cells transformed with the Spt5 pET vector reproducibly caused slow growth, suggesting that expression of human Spt5 is toxic to E. coli. The resulting loss of the expression plasmid was minimized by maintaining ampicillin selection with several fresh medium changes during growth. This allowed a significant increase in the final level of expressed protein. The majority of both the Spt4 and Spt5 proteins produced in E. coli was insoluble, but both proteins were solubilized in the presence of 6 m urea. Each subunit was individually purified over a nickel column in the presence of urea. The purified subunits were renatured together by dialysis, and the soluble fraction was loaded onto a Mono Q column. Free Spt4 flowed through the column and the DSIF heterodimer eluted at 400 mm KCl (Fig. 2 B). To obtain the complete NELF protein, NELF-E, the smallest subunit, was FLAG-tagged at its N terminus and stably transfected into HeLa S3 cells. Anti-FLAG M2 antibodies were used to affinity-purify NELF-E-containing complexes from both cytoplasmic and nuclear fractions as described under “Experimental Procedures” and outlined in Fig. 3 A. The majority of the NELF was present in the cytoplasmic fraction, and no difference was seen in the properties of NELF isolated from either the cytoplasmic or nuclear fraction (data not shown). Because of an excess of the apparent 52-kDa NELF-E subunit in the isolated material, the NELF complex was further purified by chromatography on Mono Q (Fig. 3 B). NELF eluted at 360 mm KCl. Under the conditions used, the intact NELF complex separated from free NELF-E and a few other proteins that associated with the NELF complex during affinity purification. The presence of 0.1% Nonidet P-40 throughout purification increased NELF activity and also prevented precipitation of the protein during purification and storage at −80 °C. Although the original purification of the NELF complex reported the presence of five subunits co-eluting with NELF activity (27Yamaguchi 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 (622) Google Scholar), recent protein sequence analysis indicates that the same gene encodes two of these subunits, NELF-C and NELF-D. 2T. Wada, Y. Yamaguchi, and H. Handa, manuscript in preparation. As seen in Fig. 3 B, purification of NELF on Mono Q partially resolves two different complexes that differ in the form of the NELF-C/D subunit with the complex containing the lower mobility C-form eluting first. This resolution of the two forms may not have been observed in the original purification of NELF, because the Mono Q column was used at an early stage in the purification before the subunit composition could be seen. We tested the activity of the two NELF fractions, 31 and 32, that differ in NELF-C/D composition but found no differences in activity in our add-back transcription assay (data not shown). In an attempt to reconstitute the negative elongation activity observed with HNE, the effect of adding purified DSIF and purified NELF to isolated early elongation complexes was tested (Fig. 4). Transcripts in the isolated complexes were elongated for 5 min in the presence of 0, 6, 13, 25, 50, or 100 ng of DSIF and 5 ng of NELF or with 0, 0.6, 1.3, 2.5, 5, or 10 ng of NELF and 50 ng of DSIF. At the concentrations used in this assay, DSIF or NELF added individually had no observable effects on the elongation rate. When both of these factors were added together, a negative effect was observed on approximately half of the polymerases (Fig. 4). We were unable to find conditions in which all polymerases were affected by DSIF and NELF (see “Discussion”). Using saturating concentrations of DSIF and NELF, polymerases that were affected by these factors elongated at one-third the rate of the unaffected polymerases. A maximal reduction in the rate of DSIF and NELF affected polymerases occurred when ∼50 ng of DSIF and 5 ng of NELF were added (Fig. 4 and data not shown). Increasing the concentrations of DSIF or NELF above these values did not affect the percentage of polymerases that were affected by these factors. When DSIF was added at a 10-fold higher concentration than the highest concentration tested in Fig. 4, all polymerases increased their rate of elongation by ∼20% (data not shown). This stimulatory activity was independent of NELF. Concentrations of NELF up to 15-fold higher than those used here had no effect on the elongating polymerase in the absence of DSIF. Because independent purifications resulted in DSIF preparations that had specific activities that differed by up to a factor of 5, it is likely that only a fraction of the DSIF in any of these samples was active. In contrast, separate purifications of NELF had very similar specific activities (data not shown). To determine whether the negative activity of DSIF and NELF could be reversed, increasing amounts of P-TEFb (0, 25, 50, 100, or 200 ng) were added back to isolated elongation complexes in reactions containing 50 ng of DSIF and 5 ng of NELF. As the level of P-TEFb increased, the ability of the polymerase to generate longer transcripts was restored (Fig. 5). The transcripts generated with the highest level of P-TEFb were on average slightly shorter than those seen in the absence of any added factors. This was not changed by the addition of even higher levels of P-TEFb (data not shown). This would be expected if the hig
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