Transcription initiation as catalyzed by T7 RNA polymerase consists primarily of promoter binding, strand separation, nucleotide binding, and synthesis of the first phosphodiester bond. The promoter strand separation process occurs at a very fast rate, but promoter opening is incomplete in the absence of the initiating NTPs. In this paper, we investigate how initiating NTPs affect the kinetics and thermodynamics of open complex formation. Transient state kinetic studies show that the open complex, ED(o), is formed via an intermediate ED(c), and the conversion of ED(c) to ED(o) occurs with an unfavorable equilibrium constant. In the presence of the initiating NTP that base-pairs with the template at position +2, the process of open complex formation is nearly complete. Our studies reveal that the nucleotide that drives open complex formation needs to be a triphosphate and to be correctly base-paired with the template. These results indicate that the melted template DNA in the open complex is positioned to bind the +2 NTP. The addition of +1 NTP alone does not stabilize the open complex; nor is it required for +2 NTP binding. However, there appears to be cooperativity in initiating NTP binding in that the binding of +2 NTP facilitates +1 NTP binding. The dissection of the initiation pathway provides insights into how open complex formation steps that are sensitive to the promoter sequence upstream from the initiation start site modulate the affinity of initiating NTPs and allow transcription initiation to be regulated by initiating NTP concentration.
Transcription initiation by T7 RNA polymerase (T7 RNAP) is regulated by the specific promoter DNA sequence that is classically divided into two major domains, the binding domain (−17 to −5) and the initiation domain (−4 to +6). The occurrence of nonconsensus bases within these domains is responsible for the diversity of promoter strength, the basis of which was investigated by studying T7 promoters with changes in the promoter specificity region (−13 to −6) of the binding domain and/or the melting region (−4 to −1) of the initiation domain. The transient state kinetics and thermodynamic studies revealed that multiple steps in the pathway of transcription initiation are modulated by the promoter DNA sequence. Three base changes in the promoter specificity region at −11, −12, and −13, found in the natural φ3.8 promoter, reduced the overall affinity of the T7 RNAP for the promoter DNA by 2−3-fold and decreased the rate of pppGpG synthesis, the first RNA product. Promoter opening is thermodynamically driven in T7 RNAP, and a single base change in the melting region (TATA to TAAA) decreased the extent of open complex generated at equilibrium. This base change in the melting region also increased the Kd of (+1) GTP and the dissociation rate of pppGpG. Thus, transcription initiation at various T7 promoters is differentially regulated by initiating GTP concentration. The specificity and melting regions of T7 promoter DNA act both independently and synergistically to affect distinct steps of transcription initiation. Although each step in the initiation pathway is affected to a small degree by promoter sequence variations, the cumulative effect dictates the overall promoter strength.
Bacteriophage T7 lysozyme binds to T7 RNA polymerase and inhibits transcription initiation and the transition from initiation to elongation. We have investigated each step of transcription initiation to determine where T7 lysozyme has the most effect. Stopped flow and equilibrium DNA binding studies indicate that T7 lysozyme does not inhibit the formation of the preinitiation open complex (open complex in the absence of initiating nucleotide). T7 lysozyme, however, does prevent the formation of a fully open initiation complex (open complex in the presence of the initiating nucleotide). This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP Kd is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from -4 to +2). Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex. As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2- to 5-mer that delays the transition from the initiation to the elongation phase. Bacteriophage T7 lysozyme binds to T7 RNA polymerase and inhibits transcription initiation and the transition from initiation to elongation. We have investigated each step of transcription initiation to determine where T7 lysozyme has the most effect. Stopped flow and equilibrium DNA binding studies indicate that T7 lysozyme does not inhibit the formation of the preinitiation open complex (open complex in the absence of initiating nucleotide). T7 lysozyme, however, does prevent the formation of a fully open initiation complex (open complex in the presence of the initiating nucleotide). This is consistent with the results that in the presence of T7 lysozyme the rate of G ladder RNA synthesis is about 5-fold slower and the GTP Kd is about 2-fold higher, but T7 lysozyme does not inhibit the initial rate of RNA synthesis with a premelted bulge-6 promoter (bubble from -4 to +2). Neither the RNA synthesis rate nor the extent of promoter opening is restored by increasing the initiating nucleotide concentration, indicating that T7 lysozyme represses transcription by interfering with the formation of a stable and a fully open initiation bubble or by altering the structure of the DNA in the initiation complex. As a consequence of the unstable initiation bubble and/or the inhibition of the conformational changes in the N-terminal domain of T7 RNAP, T7 lysozyme causes an increased production of abortive products from 2- to 5-mer that delays the transition from the initiation to the elongation phase. The regulation of transcription in bacteriophage T7 RNA polymerase (RNAP) 1The abbreviations used are: RNAP, RNA polymerase; 3′-dGTP, 3′-deoxyguanosine-triphosphate; ds, double-stranded; 2-AP, 2-aminopurine; nt, nontemplate; t, template. as in all RNA polymerases is dependent on the efficiency of each step of transcription, such as binding promoter DNA, binding nucleotides, overcoming abortive synthesis, RNA synthesis, and termination. Each of these steps is therefore a potential target for regulation. T7 transcription regulation begins with the gradual entry of the phage DNA into a host Escherichia coli cell (1McAllister W.T. Morris C. Rosenberg A.H. Studier F.W. J. Mol. Biol. 1981; 153: 527-544Crossref PubMed Scopus (82) Google Scholar) followed by the ability of the T7 RNAP, without the aid of auxiliary factors, to recognize and transcribe the phage promoters (2McAllister W.T. Cell Mol. Biol. Res. 1993; 39: 385-391PubMed Google Scholar, 3McAllister W.T. Nucleic Acids Mol. Biol. 1997; 11: 15-25Crossref Google Scholar). The phage then makes T7 lysozyme, which represses transcription by T7 RNA polymerase. Unlike the LacI or the LexA repressors, which sterically block promoters from their polymerases, T7 lysozyme binds the T7 RNAP, not the DNA, forming a tertiary complex with the polymerase and DNA (4Schlax PJ. Capp M.W. Record M.T. J. Mol. Biol. 1995; 245: 331-350Crossref PubMed Scopus (101) Google Scholar, 5Bertrand-Burggraf E. Hurstel S. Daune M. Schnarr M. J. Mol. Biol. 1987; 193: 293-302Crossref PubMed Scopus (59) Google Scholar, 6Zhang X. Studier F.W. J. Mol. Biol. 1997; 269: 10-27Crossref PubMed Scopus (62) Google Scholar, 7Kumar A. Patel S.S. Biochemistry. 1997; 36: 13954-13962Crossref PubMed Scopus (26) Google Scholar). T7 lysozyme binds to the T7 RNAP at a site distal to the polymerase active site as confirmed by both biochemical data and a crystal structure of the T7 RNAP-T7 lysozyme complex (8Jeruzalmi D. Steitz T.A. EMBO J. 1998; 17: 4101-4113Crossref PubMed Scopus (154) Google Scholar, 9Moffatt B.A. Studier F.W. Cell. 1987; 49: 221-227Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 10Cheng X. Zhang X. Pflugrath J.W. Studier F.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4034-4038Crossref PubMed Scopus (203) Google Scholar). Previous studies have shown that T7 lysozyme inhibits transcription initiation and promoter clearance but not elongation (6Zhang X. Studier F.W. J. Mol. Biol. 1997; 269: 10-27Crossref PubMed Scopus (62) Google Scholar, 7Kumar A. Patel S.S. Biochemistry. 1997; 36: 13954-13962Crossref PubMed Scopus (26) Google Scholar). However, the exact mechanism by which these processes are inhibited is not known. We have therefore investigated T7 lysozyme inhibition under pre-steady state conditions enabling us to more fully dissect the steps that are altered during transcription. Transcription initiation occurs with a minimum of three steps (Reaction 1). In the first step, RNAP (E) binds to the promoter DNA (D) to form a closed complex, EDc, which isomerizes to form the preinitiation open complex, EDo. The preinitiation open complex binds the initiating nucleotides (N) to form the initiation complex, EDoNN. E+D⇄EDC⇄EDo⇄EDoNNReaction 1 A stopped flow kinetic study of DNA binding showed that T7 lysozyme has a very small effect on the observed rate of formation of the preinitiation open complex. In the presence of T7 lysozyme, the initial RNA synthesis rate is slow even with saturating initiating nucleotide. The 2-AP fluorescence of the DNA indicates that the promoter in the initiation complex is not fully open. This is consistent with the results that T7 lysozyme affects the rate of initiation on a duplex promoter but not on a permanently open promoter DNA or a promoter DNA with even a single mismatch in the initiation region. The destabilization or the alteration of the structure of the initiation complex by T7 lysozyme is responsible for the inhibition of initial RNA synthesis and could also be the cause for the formation of more abortive products, which in turn delays the transition from initiation to elongation. Synthetic DNA and Other Materials—The oligodeoxynucleotides (unmodified and 2-AP-modified) were synthesized by Integrated DNA Technologies (Coralville, IA) and supplied as desalted samples. As described previously (11Stano N.M. Patel S.S. J. Mol. Biol. 2002; 315: 1009-1025Crossref PubMed Scopus (26) Google Scholar), the oligodeoxynucleotides were further purified by polyacrylamide gel electrophoresis, electroelution, and ethanol precipitation. The 3′-dGTP was purchased from TriLink Biotechnologies (San Diego, CA). Protein—T7 RNAP was overexpressed in E. coli BL21/pAR1219 (12Davanloo P. Rosenberg A.H. Dunn J.J. Studier F.W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2035-2039Crossref PubMed Scopus (731) Google Scholar). The enzyme was purified as described previously (13Jia Y.P. Kumar A. Patel S.S. J. Biol. Chem. 1996; 271: 30451-30458Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Jia Y. Patel S.S. J. Biol. Chem. 1997; 272: 30147-30153Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Jia Y. Patel S.S. Biochemistry. 1997; 36: 4223-4232Crossref PubMed Scopus (95) Google Scholar) with the exception that the CM-Sephadex separation step was eliminated. The purified enzyme was stored at -80 °C in 20 mm sodium phosphate, pH 7.7, 1 mm trisodium EDTA, 1 mm dithiothreitol, 100 mm sodium chloride, and 50% (v/v) glycerol. The enzyme concentration was calculated from its absorbance at 280 nm and with a molar extinction coefficient of 1.4 × 105m-1 cm-1 (16King G.C. Martin C.T. Pham T.T. Coleman J.E. Biochemistry. 1986; 25: 36-40Crossref PubMed Scopus (90) Google Scholar). T7 lysozyme was purified according to a reported procedure (10Cheng X. Zhang X. Pflugrath J.W. Studier F.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4034-4038Crossref PubMed Scopus (203) Google Scholar). The purified enzymes were checked for the lack of DNA exonuclease activity. Pre-steady State Kinetics of RNA Synthesis—The pre-steady state kinetic experiments were carried out on a rapid chemical quenched flow instrument (KinTek Corp., Austin, TX). As a general protocol, T7 RNAP and DNA preincubated either in the presence or absence of T7 lysozyme in buffer X (50 mm Tris acetate, pH 7.5, 100 mm sodium acetate, 10 mm magnesium acetate, 5 mm dithiothreitol) were mixed rapidly with NTPs containing [γ-32P]GTP in buffer L (50 mm Tris acetate, pH 7.5, 10 mm magnesium acetate, 5 mm dithiothreitol). Additional magnesium acetate was added to the NTP solution to maintain a constant amount of free Mg2+. The temperature was maintained at 25 °C using a water bath in all pre-steady state experiments. After predetermined time intervals, the reaction was quenched by rapidly mixing 1 n HCl from a third syringe. Chloroform was then added, and the reactions were neutralized by the addition of base (0.25 m Tris base and 1 m sodium hydroxide). The RNA products were resolved by electrophoresis on a highly cross-linked (23% polyacrylamide, 3% bis-acrylamide, 3 m urea) gel at 55 °C (110 W) on a Bio-Rad sequencing gel apparatus (0.25-mm spacers and comb). The gels were exposed to a phosphor screen and scanned on a Typhoon instrument (Molecular Dynamics), and the RNA products were quantified using the ImageQuaNT program. Stopped Flow Kinetics—The stopped flow experiments were carried out at 25 °C using a SF-2001 spectrophotometer from KinTek Corp (Austin, TX) equipped with a photomultiplier detection system. The kinetics of GTP binding was monitored by mixing increasing concentrations of GTP in buffer A (50 mm Tris acetate, pH 7.5, 50 mm sodium acetate, 10 mm magnesium acetate, 5 mm dithiothreitol) from one syringe with a preincubated solution of T7 RNAP and dsDNA with or without T7 lysozyme in buffer A from the second syringe. The dsDNA contained a single 2-AP residue at position nt(+4) on the template strand. Additional magnesium acetate was included with the GTP solution to maintain a constant concentration of free Mg2+. As the two solutions (30 μl from each syringe) were rapidly mixed (flow rate of 6.0 ml s-1), the 2-AP was excited at 315 nm. The progress of the reaction was monitored by measuring the intensity of the fluorescence emission using a cut-on filter >360 nm (WG360, Hi-Tech Scientific, serial no. 273129). Multiple traces (3McAllister W.T. Nucleic Acids Mol. Biol. 1997; 11: 15-25Crossref Google Scholar, 4Schlax PJ. Capp M.W. Record M.T. J. Mol. Biol. 1995; 245: 331-350Crossref PubMed Scopus (101) Google Scholar, 5Bertrand-Burggraf E. Hurstel S. Daune M. Schnarr M. J. Mol. Biol. 1987; 193: 293-302Crossref PubMed Scopus (59) Google Scholar, 6Zhang X. Studier F.W. J. Mol. Biol. 1997; 269: 10-27Crossref PubMed Scopus (62) Google Scholar, 7Kumar A. Patel S.S. Biochemistry. 1997; 36: 13954-13962Crossref PubMed Scopus (26) Google Scholar, 8Jeruzalmi D. Steitz T.A. EMBO J. 1998; 17: 4101-4113Crossref PubMed Scopus (154) Google Scholar, 9Moffatt B.A. Studier F.W. Cell. 1987; 49: 221-227Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 10Cheng X. Zhang X. Pflugrath J.W. Studier F.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4034-4038Crossref PubMed Scopus (203) Google Scholar) were averaged to optimize the signal. The KinTek stopped flow kinetic software was used to fit the stopped flow kinetic traces to Equation 1 describing single or multiple exponential changes, F=∑An×exp(-kobs,n×t)+C(Eq. 1) where F is the fluorescence intensity at time t; n is the number of exponential terms; An and kobs,n are the amplitude and the observed rate constant of the nth term, respectively; and C is the fluorescence intensity at t = 0. The observed rate constant (kobs) was plotted as a function of GTP, and the dependence was fit by nonlinear regression analysis to the hyperbolic equation 2 (17Johnson K.A. Sigman D.S. The Enzymes. Academic Press, Inc., New York1992: 1-61Google Scholar) using SigmaPlot. kobs=kconf×[GTP]Kd+[GTP](Eq. 2) Where kconf is the rate of a conformational change upon GTP binding, and Kd is the equilibrium dissociation constant of GTP. Equilibrium Fluorescence Measurements—T7 RNAP (1 μm) was mixed with 0.5 μm DNA containing a single 2-AP residue at t(-4) in Buffer A in a 200-μl quartz cuvette at 25 °C. The sample was excited with 315-nm light (2.5-nm bandwidth), and the resulting fluorescence intensity at 370 nm (2.5-nm bandwidth) was measured on a Fluoro-Max-2 spectrofluorometer (Jobin Yvon-Spex Instruments S.A., Inc.) using the DataMax software program. The fluorescence was then measured after the addition of T7 lysozyme (10 μm). Similar experiments were carried out in the presence of +2 NTP (500 μm 3′-dGTP, 1 mm ATP, or 4 mm GTP). Additional magnesium acetate was included with the GTP (4 mm) to maintain a constant amount of free Mg2+ in solution. The fluorescence of T7 RNAP and T7 lysozyme was subtracted out by carrying out a control experiment with the nonfluorescent DNAs. The corrected fluorescence is F = Ff - Fnf where Ff and Fnf are the fluorescence intensities of samples containing 2-AP-modified and nonfluorescent DNA, respectively. Promoter DNAs—Several synthetic promoter DNAs were used in these studies (Table I). The dsDNA is a fully duplex promoter that contains the T7 ϕ10 promoter consensus sequence from -21 to +19 and initiates RNA synthesis at +1 with the sequence GGG. The p-dsDNA and bulge-6 DNAs are considered mimics of an opened promoter. To monitor open complex formation and nucleotide binding the fluorescent adenine analog 2-AP was incorporated at position -4 in the template or at position +4 in the nontemplate strand relative to the transcription start site. Altered promoters with the initiation sequence (GAC) were used to distinguish binding of the +1 and +2 initiating nucleotides.Table ISequence of the DNA promotersA, 2-aminopurine.DNADescriptionSequencep-dsDNA-4 to +19 ss template5′-AAATTAATACGACTCAC-3′3′-TTTAATTATGCTGAGTGATATCCCTCTGGTGTTGCCAAAG-5′p-dsDNA(GAC)-4 to +19 ss template5′-AAATTAATACGACTCAC-3′3′-TTTAATTATGCTGAGTGATATCtgTCTGGTGTTGCCAAAG-5′Bulge-6-4 to +2 mismatch5′-AAATTAATACGACTCACCCGCATGAGACCACAACGGTTTC-3′3′-TTTAATTATGCTGAGTGATATCCCTCTGGTGTTGCCAAAG-5′Bulge-1-2 AA mismatch5′-AAATTAATACGACTCACTAAAGGGAGACCACAACGGTTTC-3′3′-TTTAATTATGCTGAGTGATATCCCTCTGGTGTTGCCAAAG-5′dsDNA40-bp concensus5′-AAATTAATACGACTCACTATAGGGAGACCACAACGGTTTC-3′3′-TTTAATTATGCTGAGTGATATCCCTCTGGTGTTGCCAAAG-5′dsDNA(GAC)+2 and +3 bases altered from consensus5′-AAATTAATACGACTCACTATAGacAGACCACAACGGTTTC-3′3′-TTTAATTATGCTGAGTGATATCtgTCTGGTGTTGCCAAAG-5′ Open table in a new tab T7 Lysozyme Does Not Affect Promoter Binding or the Rate of Preinitiation Open Complex Formation—Previous studies have shown that T7 lysozyme inhibits both transcription initiation and the transition from initiation to elongation (7Kumar A. Patel S.S. Biochemistry. 1997; 36: 13954-13962Crossref PubMed Scopus (26) Google Scholar). T7 lysozyme does not reduce transcription by preventing the promoter DNA from binding to the T7 RNAP or by decreasing the affinity of the promoter for the RNAP (6Zhang X. Studier F.W. J. Mol. Biol. 1997; 269: 10-27Crossref PubMed Scopus (62) Google Scholar, 7Kumar A. Patel S.S. Biochemistry. 1997; 36: 13954-13962Crossref PubMed Scopus (26) Google Scholar, 18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar). Because this was determined using indirect method, we sought to verify the observation with a more direct assay. The kinetics of promoter binding and the formation of the preinitiation open complex were measured in real time using a promoter DNA that was modified with the fluorescent adenine analog 2-AP (Table I). During open complex formation, the -4 to +2/+3 region of the promoter is converted from a duplex to a single-stranded region. If 2-AP is substituted for the adenines in the melted region, open complex formation is accompanied by an increase in 2-AP fluorescence, which can be easily monitored. Any adenine in the melting region may be substituted with 2-AP. The greatest increase in fluorescence upon binding to T7 RNAP is observed when the 2-AP is positioned at the -4 position of the template strand, t(-4) (19Bandwar R.P. Patel S.S. J. Biol. Chem. 2001; 276: 14075-14082Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This is because the adenine at t(-4) undergoes a large structural change, becoming both unpaired and unstacked from its neighboring guanine (20Cheetham G.M.T. Jeruzalmi D. Steitz T.A. Nature. 1999; 399: 80-83Crossref PubMed Scopus (275) Google Scholar), and it is the latter process that gives the large fluorescence increase (21Cheetham G.M.T. Steitz T.A. Science. 1999; 286: 2305-2309Crossref PubMed Scopus (292) Google Scholar). We have verified that the insertion of 2-AP does not affect promoter binding or transcription (13Jia Y.P. Kumar A. Patel S.S. J. Biol. Chem. 1996; 271: 30451-30458Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Jia Y. Patel S.S. J. Biol. Chem. 1997; 272: 30147-30153Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The steps of DNA binding and the formation of the preinitiation open complex were measured with the dsDNA promoter containing 2-AP at t(-4), as described previously (19Bandwar R.P. Patel S.S. J. Biol. Chem. 2001; 276: 14075-14082Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). T7 RNAP in the presence or the absence of T7 lysozyme was mixed with the promoter DNA in a stopped flow apparatus, and the increasing fluorescence intensity was monitored as a function of time. We used 12 μm of T7 lysozyme, which is above the Kd of T7 lysozyme (7Kumar A. Patel S.S. Biochemistry. 1997; 36: 13954-13962Crossref PubMed Scopus (26) Google Scholar), as higher concentrations increased the background fluorescence interfering with signal detection. The hyperbolic fit of the observed rate versus [DNA] provided K½ and an observed rate of preinitiation open complex equal to 0.9 ± 0.2 μm and 116 ± 6 s-1 and 1.1 ± 0.5 μm and 87 ± 10 s-1 in the absence and presence of T7 lysozyme, respectively (see supplemental material). The similar rates of DNA binding with and without T7 lysozyme indicates that T7 lysozyme has very little effect on the rate of preinitiation open complex formation. T7 Lysozyme Decreases the Pre-steady State Rate of RNA Synthesis but Does Not Significantly Affect the Kd of the Initiating Nucleotide—Subsequent to the steps of promoter DNA binding and the formation of a preinitiation open complex are the steps of initiating nucleotide binding to form the initiation complex that is ready to make the RNA. By monitoring the steady state kinetics of abortive synthesis, it has been determined that the Km of the initiating nucleotides GTP increases in the presence of T7 lysozyme (18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar). The steady state Km, however, is a kinetic constant whose value is influenced by the formation as well as the dissociation rates of the abortive products and thus may not reflect the Kd of GTP, which may be greater than, less than, or equal to the Km (17Johnson K.A. Sigman D.S. The Enzymes. Academic Press, Inc., New York1992: 1-61Google Scholar). Therefore, pre-steady state kinetic experiments were used to measure the rate of RNA synthesis with increasing [GTP] with and without T7 lysozyme. The dsDNA promoter was preincubated with T7 RNAP in the presence or the absence of T7 lysozyme, and transcription was initiated by the rapid addition of [γ-32P]GTP. The reactions were quenched after millisecond time intervals with the aid of a rapid chemical quenched flow apparatus, and the RNA products were quantitated. In the presence of GTP alone, we see the production of pppGpG and pppGpGpG RNA products and also some pppGpGpGpG at longer times (Fig. 1, left panel). The G ladder production increased linearly with time up to 0.25 s, and the slope provided the initial rate. The initial rate was plotted as a function of [GTP], and the results indicated that the rate at maximal [GTP] is reduced ∼5-fold (from 7 s-1 to 1.5 s-1) when T7 lysozyme is present (Fig. 1, right panel). Because of the low signal, we were unable to get an accurate value of the GTP Kd from this radiometric assay in the presence of T7 lysozyme. A 20-fold reduction in catalytic efficiency was calculated from the initial slope of rate versus [GTP] dependence (11.6 ± 2.5 s-1 mm-1 without T7 lysozyme and 0.56 ± 0.1 s-1 mm-1 in the presence of T7 lysozyme). To determine the effect of T7 lysozyme on the Kd of the initiating GTP, a more sensitive fluorescence assay was used. In this assay, GTP binding and a subsequent conformational change is monitored by following the changes in the 2-AP-modified promoter DNA (14Jia Y. Patel S.S. J. Biol. Chem. 1997; 272: 30147-30153Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Bandwar R.P. Jia Y. Stano N.M. Patel S.S. Biochemistry. 2002; 41: 3586-3595Crossref PubMed Scopus (32) Google Scholar). Using a stopped flow apparatus, T7 RNAP and the dsDNA containing 2-AP at nt(+4) with or without T7 lysozyme was rapidly mixed with a solution of GTP (Fig. 2a). This resulted in a time-dependent increase in fluorescence (Fig. 2b), and the observed rate was plotted against GTP concentration (Fig. 2c). The hyperbolic fit provided average Kd values of the +1 and +2 GTP as 435 ± 276 and 742 ± 233 μm in the absence and the presence of T7 lysozyme, respectively. Note that in the absence of T7 lysozyme, the GTP Kd is consistent with the values previously reported (11Stano N.M. Patel S.S. J. Mol. Biol. 2002; 315: 1009-1025Crossref PubMed Scopus (26) Google Scholar, 22Bandwar R.P. Jia Y. Stano N.M. Patel S.S. Biochemistry. 2002; 41: 3586-3595Crossref PubMed Scopus (32) Google Scholar). Thus, T7 lysozyme doubles the apparent Kd of GTP and causes over a 3-fold decrease in the maximal rate of a conformational change occurring upon GTP binding (kconf). T7 Lysozyme Does Not Inhibit the Rate of Initial RNA Synthesis on a "Premelted" Promoter—The experiments thus far show that T7 lysozyme inhibits the rate of RNA synthesis during initiation. Both the stopped flow and radiometric assays show that the inhibition cannot be overcome by increasing the initiating GTP concentration. We next measured transcription in the presence of all rNTPs to observe the effect of T7 lysozyme on the synthesis of longer RNA. T7 RNAP and dsDNA in the absence or the presence of T7 lysozyme was mixed with four rNTPs (1 mm of GTP and 500 μm other NTPs) and [γ-32P]GTP in a rapid chemical quenched flow instrument. The reaction was quenched after various times, and the products resolved on a sequencing gel are shown in Fig. 3a. In the presence of T7 lysozyme, the pre-steady state rate of RNA synthesis is 3-fold slower (Table II), which is evident from the decrease in the initial burst phase with T7 lysozyme shown in Fig. 3b. The abortive products from 2- to 5-mer on the other hand are produced in greater amounts with T7 lysozyme (Fig. 3c). Hence, the steady state rate of RNA synthesis is actually higher in the presence of T7 lysozyme (Table II). The 19-mer run-off product is produced with a longer delay in the presence of T7 lysozyme (Fig. 3d).Table IIKinetic parameters of RNA synthesis with and without T7 lysozymePromoterPre-steady state rate of RNA synthesisSteady-state rate of RNA synthesis—T7 lysozyme+T7 lysozyme—T7 lysozyme+T7 lysozymeμM s-1μM s-1dsDNA12 ± 54 ± 0.61.0 ± 0.052 ± 0.1Bulge-614.5 ± 1.711 ± 0.24 ± 0.46 ± 0.2Bulge-112 ± 1.214 ± 0.62 ± 0.37 ± 0.6 Open table in a new tab Similar measurement of the pre-steady state kinetics of RNA synthesis on a "premelted" DNA was carried out to determine whether T7 lysozyme inhibits the chemistry step. Bulge-6 is considered a mimic of a melted promoter because it contains 6 noncomplementary bases to the template strand in the initiation region (-4 to +2). This substrate was chosen over the p-dsDNA because the bulge-6 contains the nontemplate strand, which is capable of interacting with T7 RNAP. The initial rate of total RNA synthesis with the bulge-6 promoter is unaffected by the presence of T7 lysozyme (Table II), as evident from the similar burst phase with and without T7 lysozyme in Fig. 4a. The bulge-1 DNA shows a similar behavior as bulge-6 (Fig. 4d). This indicates that T7 lysozyme does not alter the chemical step of RNA synthesis as long as the DNA is premelted or easily melted. Even though T7 lysozyme had little effect on the RNA synthesis rate, the production of abortive products from 2- to 5-mer with bulge-6 and from 2- to 5-mer with bulge-1 was increased in the presence of T7 lysozyme (Fig. 4, b and e). This results in a higher steady state rate with T7 lysozyme (Table II). T7 Lysozyme Affects the Formation of the Open Complex in the Presence of the Initiating Nucleotide—Our results indicate that the decrease in the rate of initiation cannot be attributed to a defect in the formation of the preinitiation complex or a defect in chemistry or solely to an increase in the GTP Kd. Increased abortive synthesis in the presence of T7 lysozyme appears to be due to an effect on the stability of the open complex during initiation. To explore this idea, we have used 2-AP fluorescence to investigate the nature of the open complexes in the preinitiation and initiation stages with and without T7 lysozyme. The crystal structure of T7 RNAP-promoter complex, which likely represents the structure of the preinitiation complex, shows that the promoter DNA is unpaired from -4 to +2 and that the -4 base is unstacked from the -5 base (21Cheetham G.M.T. Steitz T.A. Science. 1999; 286: 2305-2309Crossref PubMed Scopus (292) Google Scholar). A promoter DNA containing 2-AP at t(-4) when bound to T7 RNAP has a higher fluorescence relative to the fluorescence of the free 2-AP-modified DNA, mainly because the t(-4) base is unstacked in the open complex. To investigate the nature of the open complex with and without T7 lysozyme, we compared the 2-AP fluorescence of E ·p-dsDNA and E ·dsDNA (23Stano N.M. Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 37292-37300Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The fluorescence of E·dsDNA is 30% of E·p-dsDNA (Fig. 5a) as measured with the (GAC) promoter in the absence of the initiating nucleotide. The fluorescence of E·dsDNA·ATP, however, is close to that of E·p-dsDNA·ATP, indicating as previously reported that the addition of the +2 nucleotide NTP drives open complex formation (23Stano N.M. Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 37292-37300Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Even in the presence of T7 lysozyme (L), the fluorescence of EL·dsDNA is 30% of the EL·p-dsDNA. Thus, T7 lysozyme has little effect on the formation or the stability of the preinitiation open complex. However, the fluorescence of EL·dsDNA·ATP is only 42% of EL·p-dsDNA·ATP. The addition of GTP, the +1 nucleotide, along with the ATP produced no further increase in fluorescence (data not shown). The inability of the +2 ATP to drive open complex formation to completion in the presence of T7 lysozyme was not due to subsaturating concentrations of ATP. The Kd of the +2 nucleotide was obtained from fluorescence equilibrium titrations. The preinitiation E·dsDNA complex of the (GAC) promoter in the absence or the presence of T7 lysozyme was titrated with ATP, and the increase in fluorescence was measured. The ATP dependence fit to a hyperbolic equation provided an ATP Kd of 89 ± 12 μm in the absence and 134 ± 64 μm in the presence of T7 lysozyme (data not shown). The +2 nucleotide Kd is only slightly affected by T7 lysozyme. The fact that the fluorescence of EL·dsDNA·ATP does not reach the level of the fully open promoter even with a high concentration of the initiating nucleotide, indicates that T7 lysozyme affects the structure of the initiation complex rather than the equilibrium constant of the initiation complex. Similar experiments were carried out with the (GGG) promoter to eliminate the possibility that the effect of T7 lysozyme on the initiation complex was due to the use of the nonconsensus (GAC) promoter. As shown in Fig. 5b, the results are similar to that of the (GAC) promoter. The nucleotide 3′-dGTP was substituted for GTP to avoid complications from RNA synthesis (14Jia Y. Patel S.S. J. Biol. Chem. 1997; 272: 30147-30153Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22Bandwar R.P. Jia Y. Stano N.M. Patel S.S. Biochemistry. 2002; 41: 3586-3595Crossref PubMed Scopus (32) Google Scholar, 23Stano N.M. Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 37292-37300Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The results indicate that the preinitiation open complex is not affected by T7 lysozyme, but the initiation complex is perturbed by T7 lysozyme. Because the 2-mer RNA synthesis rate was not affected by T7 lysozyme with an already or readily melted promoter, one would predict that T7 lysozyme would not affect the formation of the initiation complex with these promoters. This is indeed the case as shown in Fig. 5c. The fluorescence of bulge-6 and bulge-1 in complex with T7 RNAP is similar to that of the p-dsDNA. Upon addition of GTP, the dsDNA complex showed the characteristic increase in fluorescence from 30 to 100%, but no change was observed in the bulge DNAs. In the presence of T7 lysozyme (10 μm), a slight decrease in the fluorescence of bulge DNA preinitiation complexes was observed, but this was overcome by the addition of nucleotide. Thus, the initiation complex of bulge-6, bulge-1 or p-dsDNA was unaffected by the presence of T7 lysozyme. The fluorescence of EL·dsDNA with GTP was greater than with 3′-dGTP (65% versus 40%) most likely because of the stabilizing effects of the newly synthesized RNA. The experiments presented here were carried out to investigate the mechanism by which T7 lysozyme inhibits T7 RNAP transcription. It is already known that unlike many other transcriptional repressors, which sterically block promoters from their polymerases, T7 lysozyme does not bind DNA. Rather it directly binds to T7 RNAP to form a tertiary complex with the polymerase and DNA (7Kumar A. Patel S.S. Biochemistry. 1997; 36: 13954-13962Crossref PubMed Scopus (26) Google Scholar). More specifically, a crystal structure of the T7 lysozyme-T7 RNAP complex revealed that T7 lysozyme binds to a site distal to the polymerase active site and causes little change in the overall T7 RNAP structure with the exception of the extreme C terminus. These last four residues (FAFA883) are disordered in the complexed structure, whereas in the T7 RNAP-DNA complex structure they are located below the polymerase active site (8Jeruzalmi D. Steitz T.A. EMBO J. 1998; 17: 4101-4113Crossref PubMed Scopus (154) Google Scholar). Furthermore, biochemical data revealed that mutations in any of these four C-terminal residues result in decreased T7 RNAP activity (24Gardner L.P. Mookhtiar K.A. Coleman J.E. Biochemistry. 1997; 36: 2908-2918Crossref PubMed Scopus (33) Google Scholar) and that the C terminus is more sensitive to proteolysis in the presence of T7 lysozyme (25Huang J. Villemain J. Padilla R. Sousa R. J. Mol. Biol. 1999; 293: 457-475Crossref PubMed Scopus (34) Google Scholar). It has been proposed based on this information and steady state transcription assays that showed an increase in the NTP apparent Km during initiation that T7 lysozyme inhibits T7 RNAP by stabilizing the conformation of the T7 RNAP with an altered C terminus. This model predicts that inhibition can be overcome by the addition of nucleotide (18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar, 25Huang J. Villemain J. Padilla R. Sousa R. J. Mol. Biol. 1999; 293: 457-475Crossref PubMed Scopus (34) Google Scholar). We have further investigated this model for T7 lysozyme inhibition of T7 RNAP transcription initiation using pre-steady state techniques. During T7 RNAP transcription initiation there are several potential points for regulation by T7 lysozyme. The first potential regulatory site is promoter binding. Stopped flow experiments indicated that T7 lysozyme does not prevent T7 RNAP from binding the promoter. Similarly, the preinitiation complex formation rate was affected only to a small extent in the presence of T7 lysozyme. We next explored the possibility that T7 lysozyme altered the binding of the initiating nucleotide to the T7 RNAP-promoter preinitiation complex. A quenched flow radiometric assay measuring the rate of RNA synthesis during initiation at increasing [GTP] showed that the rate of G ladder synthesis decreased 5-fold in the presence of T7 lysozyme. The rate could not be restored even by high concentrations of nucleotide (2 mm GTP). Because obtaining a reliable GTP Kd value was impossible due to the difficulty in measuring the products synthesized in the presence of T7 lyszoyme, a fluorescent assay monitoring an increase in 2-AP fluorescence upon GTP binding was employed. The stopped flow assay also indicated that the maximal rate of a conformational change occurring upon GTP binding (kconf) was reduced ∼3-fold, whereas the average GTP Kd was increased at most 2-fold in the presence of T7 lysozyme. These results are only partly in agreement with the proposed mechanism of Villemain and Sousa (18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar) that postulated T7 lysozyme inhibition because of an increase in nucleotide Km values. According to this model, a high concentration of GTP should be able to restore the rate of RNA synthesis to uninhibited levels even in the presence of T7 lysozyme (18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar). However, the pre-steady state rate of RNA synthesis does not increase at high GTP with T7 lysozyme. The observed recovery of steady state synthesis observed by Villemain and Sousa (18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar) with increasing nucleotide may have been due to the increased rate of dissociation of small RNA products occurring during multiple turnovers, which is facilitated in the presence of T7 lysozyme (18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar). Because a defect in initiating nucleotide binding was not the sole cause for inhibition, we next examined whether T7 lysozyme inhibited the inherent ability of T7 RNAP to carry out RNA synthesis. When T7 RNAP was supplied with an already melted or an easily melted promoter (bulge-6 or bulge-1), then T7 lysozyme was unable to slow the rate of initial RNA synthesis. The lack of inhibition on the open promoter is not the result of T7 lysozyme being unable to form a tertiary complex. Complex formation is evident from the greater production of abortive products in the presence of T7 lysozyme. The result that T7 lysozyme does not hinder the ability of T7 RNAP to carry out RNA synthesis but causes additional accumulation of short RNA products is consistent with the idea that T7 lysozyme inhibits a step associated with stable open complex maintenance. The nature or the amount of the open complex formed by T7 RNAP-DNA or T7 RNAP-T7 lysozyme-DNA complexes was investigated by comparing the 2-AP fluorescence of dsDNA and p-dsDNA in complex with T7 RNAP or T7 RNAP-T7 lysozyme complex. The fluorescence of 2-AP in DNA is sensitive mainly to base-stacking interactions (26Rachofsky E.L. Osman R. Ross J.B.A. Biochemistry. 2001; 40: 946-956Crossref PubMed Scopus (312) Google Scholar); hence, the measurement of the fluorescence of 2-AP provides information about the degree of t(-4) unstacking. The fluorescence of E·dsDNA is 30% of E·p-dsDNA, and we have shown that the lower fluorescence of dsDNA is due to the unfavorable equilibrium constant for the formation of the preinitiation open complex (19Bandwar R.P. Patel S.S. J. Biol. Chem. 2001; 276: 14075-14082Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The same ratio was observed in the presence of T7 lysozyme, indicating that preinitiation open complex formation is not affected by T7 lysozyme. In the presence of the +2 NTP, the fluorescence of E·dsDNA increases to nearly the level of E·p-dsDNA (23Stano N.M. Levin M.K. Patel S.S. J. Biol. Chem. 2002; 277: 37292-37300Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In the presence of T7 lysozyme, the +2 NTP was unable to increase the fluorescence of 2-AP to the level of the p-dsDNA. This was not due to the fact that +2 NTP was not bound to the RNAP as the +2 NTP Kd is only 1.5-fold higher in the presence of T7 lysozyme. The fact that even saturating concentrations of the initiating nucleotide was unable to drive open complex formation indicates that T7 lysozyme affects the structure of the open DNA in the initiation complex rather than the equilibrium constant for open complex formation. T7 lysozyme interacts with parts of the palm, finger, and the N-terminal domain of T7 RNAP, and this mode of binding can lock the protein or affect protein flexibility to prevent a conformational change that is required to form a fully open initiation complex. There are numerous protein-DNA interactions that are required to maintain the transcription bubble and because T7 lysozyme binds at a site remote from these interactions, the action of T7 lysozyme is allosteric. The allosteric effect of T7 lysozyme results in the destabilization of the transcription bubble, which may happen because of long range perturbation of T7 lysozyme binding on the interactions of T7 RNAP with the nontemplate or the template strand. This hypothesis is consistent with the finding that the G235D and R231H mutations in the intercalating β hairpin, which is involved in maintaining the open bubble (27Brieba L.G. Sousa R. Biochemistry. 2001; 40: 3882-3890Crossref PubMed Scopus (29) Google Scholar, 11Stano N.M. Patel S.S. J. Mol. Biol. 2002; 315: 1009-1025Crossref PubMed Scopus (26) Google Scholar) have been identified as T7 lysozyme sensitive mutants (28Zhang X. T7 RNA Polymerase and T7 Lysozyme: Genetic, Biochemical, and Structural Analysis of Their Interaction and Multiple Roles in T7 Infection. 1995; (Ph.D. thesis, State University of New York at Stony Brook)Google Scholar). Similarly, the higher sensitivity of the class II ϕ3.8 promoter to T7 lysozyme (18Villemain J. Sousa R. J. Mol. Biol. 1998; 281: 793-802Crossref PubMed Scopus (21) Google Scholar) (data not shown) is consistent with the fact that the ϕ3.8 promoter is less efficiently opened by T7 RNAP than a consensus promoter (22Bandwar R.P. Jia Y. Stano N.M. Patel S.S. Biochemistry. 2002; 41: 3586-3595Crossref PubMed Scopus (32) Google Scholar). The greater production of abortive products in the presence of T7 lysozyme indicates that the RNA dissociates more frequently when T7 lysozyme is complexed to T7 RNAP. This may be because the RNA is bound less stably at the active site either because of the frequent collapse of the initiation bubble back to the duplex form or because of the disruption in the interactions of the T7 RNAP with the RNA at the active site. Nonetheless, because of the increased abortive synthesis, the RNAP spends more time in the recycling mode, which causes a delay in the transition from initiation to elongation. The fact that abortive products from 2- to 5-mer increases with T7 lysozyme also indicates that the delay in the transition from the initiation to the elongation phase could be caused by the inhibition of some or all the conformational changes of the N-terminal domain (29Tahirov T.H. Temiakov D. Anikin M. Patlan V. McAllister W.T. Vassylyev D.G. Yokoyama S. Nature. 2002; 420: 43-50Crossref PubMed Scopus (184) Google Scholar, 30Yin Y.W. Steitz T.A. Science. 2002; 298: 1387-1395Crossref PubMed Scopus (276) Google Scholar) that are necessary to make RNA products longer than 3-mer. T7 lysozyme can therefore allosterically inhibit the rotation of the N-terminal core domain or the refolding of the N-terminal domains delaying promoter clearance. We thank Dr. Rajiv Bandwar for discussions and reading of the manuscript. Download .pdf (.48 MB) Help with pdf files
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