UTP Allosterically Regulates Transcription by Escherichia coli RNA

UTP Allosterically Regulates Transcription byEscherichia coli RNA Polymerase from theBacteriophage T7 A1 Promoter

Ronald S. Johnson* and Rebecca E. Chester

Department of BiochemistryBrody School of Medicineat East Carolina UniversityGreenville, NC 27858-4354USA

In the case of Escherichia coli RNA polymerase, UTP at elevated concen-trations suppresses terminated transcript accumulation during multiple-round transcription from a DNA construct containing the T7 A1 promoterand Te terminator. The step that is affected by UTP at elevated concen-trations is promoter clearance. In an attempt to understand better themechanism by which UTP regulates this step, we analyzed the effect ofUTP on the formation of pppApU in the presence of only UTP and ATP.At elevated concentrations, UTP is a non-competitive inhibitor withrespect to ATP in the formation of pppApU. This indicates that the effectof UTP on the formation of pppApU is mediated through an allostericsite. Moreover, the magnitude of the inhibition of pppApU formation issufficient to account for the decrease in terminated transcript accumu-lation at elevated UTP concentrations. Thus, it appears that UTPmodulates terminated transcript accumulation during multiple-roundtranscription from this DNA construct by allosteric regulation of promoterclearance at the point of transcription initiation.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: Escherichia coli RNA polymerase; promoter clearance;transcription initiation; allosteric regulation; transcriptional pausing*Corresponding author

Introduction

RNA synthesis as catalyzed by Escherichia coliRNA polymerase is a complex multistep process.The first step is promoter search and open complexformation. This is followed by promoter clearancewhich involves all of the steps up to and includingthe formation of a stable ternary elongation com-plex consisting of the enzyme, the DNA templateand the nascent RNA transcript. The process ofelongation continues until a termination site isreached. At this point, the full-length RNA tran-script is released along with the enzyme. Regu-lation may occur at any step along this pathwayand may involve accessory proteins.1 – 7

In most cases, the role of nucleoside tri-phosphates in regulating the synthesis of full-length transcripts has been postulated to bemediated in a passive way by variations in theiraffinities for the active site at different pointsalong the template.4,8 – 12 To gain further insight

into the role of nucleoside triphosphates in regu-lating the synthesis of full-length transcripts, weused several DNA constructs containing eitherjust the T7 A1 promoter or the T7 A1 promoteralong with the T7 Te terminator. We investigatedthe effects of ATP and UTP on the production offull-length transcripts. These two nucleotides areinvolved in transcription initiation at the T7 A1promoter. At elevated concentrations, UTP but notATP suppressed the formation of full-length tran-scripts during multiple-round transcription fromthe DNA construct that contained the A1 promoterand the Te terminator. The data obtained here areconsistent with a model in which UTP regulatestranscription through an allosteric site.

Results

The accumulation of transcripts from the A1promoter is modulated by UTP levels duringmultiple-round transcription

The RsaI–SalI fragment from plasmid pARl707contains the A1 promoter along with the Te termi-nator from bacteriophage T7. The 50 terminal

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:[email protected]

Abbreviations used: CIP, calf intestinal alkalinephosphatase.

doi: 10.1016/S0022-2836(02)00042-6 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 318, 305–320

sequence of the transcript produced from the A1promoter is pppApUpCpG.13 During multiple-round transcription from the A1 promoter, theaccumulation of product corresponding to theterminated transcript (161 nucleotides) varied as afunction of UTP concentration (Figure l(a)). Maxi-mum accumulation of terminated transcriptsoccurred at a UTP concentration of 0.05 mM. Thisaccumulation was reduced by 40(^11)% at a UTPconcentration of 1.0 mM (Figure l(b)).

In addition to the terminated transcript, abortiveproducts were generated during this reaction time(Figure l(a)). Maximum accumulation of the 4-meroccurred at a UTP concentration of 0.5 mM andthe accumulation was reduced by 16(^4)% ata UTP concentration of 1.0 mM (Figure l(c)). Asimilar UTP concentration dependency wasobserved for the 5-mer abortive product (plot notshown). The accumulation of the 3-mer reached amaximum at a UTP concentration of 0.5 mM andremained constant (plot not shown). At anexposure time of the X-ray film appropriate foranalyses of the terminated transcript as well as the5, 4 and 3-mers, it was not possible to analyze the2-mer. In an attempt to determine the variation ofthe 2-mer as a function of UTP concentration, wedecreased the exposure time of the X-ray film.The accumulation of the 2-mer could still not be

analyzed under these conditions due to the pre-sence of a contaminant in the [g-32P]ATP thatappears to comigrate with the 2-mer product(Figure 1(d)). The intensity of the contaminantband is comparable to or less than the intensitiesof the bands corresponding to the reactionmixtures. Although this prevented a quantitativeanalysis of the 2-mer product, qualitatively itappears that the accumulation of this productreached a maximum at a UTP concentration of0.5 mM and then decreased slightly at a UTPconcentration of 1 mM. Based on these data, amechanism involving increased abortive cyclingcannot account for the decrease in the accumu-lation of terminated transcripts at elevated UTPconcentrations. Because there are no bandsbetween those corresponding to the terminatedand abortive products (Figure 1(a)), arrestedelongation complexes can also be eliminated as thecause of the suppression of terminated transcriptaccumulation at elevated UTP concentrations.

Electrophoretic analyses of terminated transcriptaccumulation as a function of time are given inFigure 2(a) (0.05 mM UTP) and (b) (1.0 mM UTP).Plots of the amount of terminated transcriptsformed as a function of time are linear over thetime span of 5–30 minutes at UTP concentrationsof 0.05 and 1.0 mM (Figure 2(c)) as well as at UTP

Figure 1. (a) Gel electrophoreticanalysis (20% (w/v) polyacryl-amide) of products formed duringmultiple-round transcription fromthe RsaI–SalI DNA fragment ofpARl707 over a time span of 30minutes as a function of UTP con-centration at 37 8C. (All electro-phoretic patterns presented herecorrespond to digital represen-tations. In each case, we have opti-mized visualization of the bandsby varying brightness and contrastsettings.) Each reaction mixturecontained 12.8 nM active enzymemolecules and 16 nM RsaI–SalIDNA fragments. The concen-trations of CTP, GTP and[g-32P]ATP (1500–3000 cpm/pmol)were each 50 mM. UTP concen-trations were varied as indicatedin the Figure. Variations in theamounts of the terminated tran-script (b) and the 4-mer product(c) formed as a function of UTPconcentration. Each point isnormalized to the value at a UTPconcentration of 0.05 mM in thecase of the terminated transcript,whereas they are normalized to thevalue at a UTP concentration of0.5 mM in the case of the 4-merproduct. Also, each point corre-

sponds to the average of three experiments. (d) Short time exposure of X-ray film showing the variation of the 2-merabortive product as a function of UTP concentration. The control contains [g-32P]ATP at the same concentration asthat used in the reaction.

306 Allosteric Regulation of E. coli RNA Polymerase

concentrations of 0.01 and 0.5 mM (data notshown). Based on promoter clearance studies thatare presented later, most of the RNA polymerasemolecules are in the recycling phase after approxi-mately five minutes. Linear plots over the timerange of 5–30 minutes indicate that most of theRNA polymerase molecules are able to recycleafter termination, that the nucleoside triphosphatesare not depleted during the course of the reactionand that there is no product inhibition. The

dependency of the relative rate of terminated tran-script accumulation on UTP concentration is illus-trated in Figure 2(d). The maximum relative ratefor terminated transcript accumulation occurred ata UTP concentration of 0.05 mM and that rate wasreduced by approximately 38% at a UTP concen-tration of 1 mM. This is consistent with the data inFigure 1(b).

The ATP concentration in the studies reported inFigures 1 and 2 was 50 mM, whereas it was 2 mMin studies that will be discussed later on the effectof UTP concentration on the formation of pppApUfrom ATP and UTP. This concentration of ATPallowed us to simplify the analysis of the UTPconcentration dependency of pppApU formation.To ascertain whether the UTP concentration depen-dency of terminated transcript accumulation isindependent of ATP concentration, we analyzedthe system at an ATP concentration of 2 mM on a20% polyacrylamide gel. For these experiments,we used [a-32P]CTP at a concentration of 50 mM tomonitor the formation of terminated transcriptsand the GTP concentration was 50 mM. Underthese conditions, maximum accumulation of termi-nated transcripts occurred at a UTP concentrationof 0.05 mM and this accumulation was reduced by23(^6)% at a UTP concentration of 1.0 mM during

Figure 2. Gel electrophoretic pattern (20% (w/v)polyacrylamide) of terminated transcript formation as afunction of time during multiple-round transcriptionfrom the RsaI–SalI DNA fragment of pAR1707 in thepresence of (a) 0.05 mM UTP and (b) 1.0 mM UTP at37 8C. The concentrations of enzyme and DNA fragmentwere the same as those given in the legend to Figure 1.The concentrations of [a-32P]CTP (750–1000 cpm/pmol),GTP and ATP were each 50 mM. (c) Variations in theformation of terminated transcripts during multiple-round transcription in the presence of 0.05 mM (X) and1.0 mM (P) UTP as a function time. The data are normal-ized to the value at 30 minutes in the presence of0.05 mM UTP. (d) Variation in the relative overall ratesfor terminated transcript accumulation as a function ofUTP concentration. The values at 0.01 and 0.5 mM UTPcorrespond to the average of two determinations andthe values at 0.05 and 1.0 mM UTP correspond to theaverage of six determinations. Each point is normalizedto the value at 0.05 mM UTP.

Figure 3. (a) Gel electrophoretic analysis (20% (w/v)polyacrylamide) of products from the RsaI–SalI DNAfragment of pAR1707 over a time span of 30 minutesduring multiple-round transcription as a function ofATP concentration at 37 8C. The concentrations ofenzyme and DNA fragment were the same as thosegiven in the legend to Figure 1. The concentrations of[a-32P]CTP (750–1000 cpm/pmol), GTP and UTP wereeach 50 mM. ATP concentrations are indicated in theFigure. (b) Variation in terminated transcript formationas a function of ATP concentration. Each point corre-sponds to the average of six independent experimentsand the data are normalized by the average of the valuesover the ATP concentration range of 0.5–2.0 mM.

Allosteric Regulation of E. coli RNA Polymerase 307

a reaction time of 30 minutes (average of fiveindependent determinations; data not shown).Although the overall trends of UTP concentrationdependencies for terminated transcript accumu-lation are the same at 0.05 and 2 mM ATP, theextent of inhibition in the presence of 2 mM ATPis less. We will address this point further in theDiscussion.

In contrast to the results obtained with UTP, ATPat elevated concentrations did not suppress theaccumulation of terminated transcripts duringmultiple-round transcription from the A1 promo-ter (Figure 3). These data provide additionalevidence that the suppression of terminated tran-script accumulation at elevated UTP concen-trations is not due to product inhibition by eitherthe terminated or abortive products. If productinhibition was the source of the variation in termi-nated transcript accumulation as a function ofUTP concentration, then elevated concentrationsof ATP should have produced the same results.Thus, although it has been reported that elevatedconcentrations of RNA inhibit transcription,14

apparently insufficient terminated transcriptswere produced to have any effect on RNAsynthesis in this case.

In all of the studies discussed above, the reac-tions were initiated by mixing solutions containingthe A1 promoter and RNA polymerase with solu-tions containing the nucleotides. Thus, the opencomplex was preformed for the first round of tran-scription. During the recycling phase, the holo-enzyme has to reassociate with the A1 promoterand form an open complex. To ascertain whetherthe results are the same when the open complex isnot preformed for the first transcriptional cycle,we initiated the reactions by mixing solutionscontaining the A1 promoter and nucleotides withsolutions containing the protein. For these studies,we used [a-32P]CTP at a concentration of 50 mM tomonitor the formation of the terminated transcript.The concentration of ATP was 2 mM, the concen-tration of GTP was 50 mM and the concentrationof UTP was varied. Under these conditions, maxi-mum accumulation of terminated transcriptsoccurred once again at a UTP concentration of0.05 mM and this accumulation was reduced by25(^3)% at a UTP concentration of 1.0 mM duringa reaction time of 30 minutes (average of threeindependent determinations; data not shown).This value is comparable to the value observedwhen the reaction was initiated by adding nucleo-tides to a solution containing the preformed opencomplex. Thus, starting with a preformed opencomplex does not alter the UTP concentrationdependency of terminated transcript accumulation.

Interaction of UTP with the RNA polymerase–A1 promoter complex as monitored in filterbinding assays

In previous filter binding studies, it was demon-strated that the A1 promoter is directly dis-placed from RNA polymerase by heparin.15,16 Weextended these studies in order to investigate theinteraction of UTP with the RNA polymerase–A1promoter complex. At elevated concentrations,UTP stabilized the RNA polymerase–A1 promotercomplex against heparin disruption (Figure 4(a)).The concentration dependency for the UTP stabili-zation of the RNA polymerase–A1 promotercomplex is illustrated in Figure 4(b). Analyses ofseveral binding isotherms yielded a value of1.3(^0.4) mM for the apparent Kd in the bindingof UTP to the RNA polymerase–A1 promotercomplex at 37 8C. These results indicate that thedecrease in terminated transcript accumulation isnot due to a UTP-mediated decrease in the stabilityof the RNA polymersae–A1 promoter complex atelevated UTP concentrations.

Interaction of UTP with RNA polymerase asmonitored by fluorescence spectroscopy

In the presence of UTP (4 mM) but the absenceof DNA, there was approximately a 62% decreasein the intensity of the fluorescence spectrum ofRNA polymerase (44 nM) at a wavelength of340 nm; however, there was no discernible shift in

Figure 4. (a) Heparin displacement of the T7 A1promoter (3.2 nM 32P-end labeled RsaI-SalI fragment ofpAR1707 at a specific radioactivity of approximately18 £ l06 cpm/nmol) from RNA polymerase (2.5 nMactive enzyme molecules) in the absence (X) andpresence (P) of UTP (2 mM) at 37 8C. The curves throughthe data correspond to exponential fits. (b) Concentrationdependency of UTP stabilization of the T7 A1 promoter–RNA polymerase complex (5 nM active enzymemolecules and 6.4 nM A1 fragment) against heparin dis-placement. Each point corresponds to the percentageincrease in the amount of complex retained on the filterrelative to that retained in the absence of UTP after aheparin challenge of 15 minutes. The curve through thedata corresponds to a hyperbolic fit.


the wavelength maximum at 340 nm (Figure 5). Inthe absence of detailed information on the locationof the tryptophan residues relative to the UTPbinding site, it is not possible to state whether thisperturbation is due entirely to a UTP-induced con-formational change in RNA polymerase or if thereis a contribution due to direct interaction of UTPwith tryptophan residues resulting in quenching.The binding isotherm obtained in the fluorometrictitration of RNA polymerase with UTP is hyper-bolic (Figure 5, inset). Analyses of several titrationsyielded a value of 0.8(^0.3) mM for the Kd of UTPbinding to RNA polymerase at 25 8C. In studiesthat were limited to UTP concentrations of lessthan 0.2 mM, Wu and Goldthwait17 observed noeffect of UTP on the fluorescence spectrum ofRNA polymerase. (No DNA was present.)Inspection of the data in the inset of Figure 5 indi-cates, in agreement with the data of Wu andGoldthwait,17 that there is little if any effect ofUTP on the fluorescence spectrum of RNA poly-merase at UTP concentrations less than 0.2 mM.

Effect of UTP on transcription elongation

In order to monitor the elongation process sepa-rate from promoter clearance, we used an elon-gation complex (A20) that was paused at positionþ20 of the transcript. Synchronously pausedternary complexes prepared by exclusion of oneor more nucleotides from the reaction mixturehave been used previously to study the elongationprocess.9,18 – 21 For this purpose, we used the RsaI–PvuII DNA fragment isolated from plasmidpAR1435. This DNA fragment contains the A1 pro-moter but not the Te terminator and is capable ofproducing a 1750 nucleotide runoff transcript. Theincorporation of [32P]CMP into RNA occurred atidentical rates at UTP concentrations of 0.05 and1.0 mM in the case of this DNA fragment (Figure6). Similar results were obtained at a UTP con-centration of 1.5 mM (data not shown). BecauseRhodes and Chamberlin22 reported that UTP atconcentrations of either 5 or 10 mM competitivelyinhibited transcription elongation in the case ofpoly[r(A–U)] synthesis, we investigated the effectof UTP at a concentration of 5 mM in the case ofthe RsaI–PvuII DNA fragment. At this concen-tration of UTP, there was a 29(^4)% decrease inthe rate of [32P]CMP incorporation (data notshown). Values of 0.5 mM,23 0.7 mM24 and1.4 mM25 have been reported for the total UTPconcentration in E. coli. Thus, although UTP atconcentrations equal to or greater than 5 mMinhibits transcription elongation, these UTP con-centrations are far greater than the reportedvalues in E. coli cells and this inhibition probablyplays no role in modulating the overall processof transcription within the cell. Up to a concen-tration of at least 1.5 mM, it appears that UTPhas little if any effect on transcription elongationin this system.

Figure 5. Emission fluorescence spectra of RNA poly-merase (44 nM total enzyme concentration) in theabsence (continuous line) and presence (dotted line) ofUTP (4 mM). Each spectrum represents the average offive scans and has been normalized to the concentrationof tryptophan residues present. The inset shows thefluorometric titration of RNA polymerase by UTP.

Figure 6. Incorporation of [32P]CMP into RNA duringelongation in the presence of 0.05 mM (X) and 1.0 mM(W) UTP, respectively, at 37 8C. The zero time point corre-sponds to the time of mixing the preformed A20 complexwith the nucleotide cocktail containing [a-32P]CTP.The concentrations of GTP, ATP and [a-32P]CTP (300–500 cpm/pmol) in the reaction mixtures were all 50 mM.

Figure 7. Gel electrophoretic analysis (8% (w/v) poly-acrylamide) of termination efficiency as a function ofUTP concentration during multiple-round transcriptionfrom the RsaI–SalI DNA fragment at 37 8C. The concen-trations of enzyme and DNA fragment were the sameas those given in the legend to Figure 1. The concen-tration of ATP was 2 mM and the concentrations of GTPand [a-32P]CTP (750–1000 cpm/pmol) were each 50 mM.(a) and (b) correspond to the same autoradiogram atdifferent brightness and contrast settings.


Effect of UTP on transcription termination

During multiple-round transcription from theRsaI–SalI fragment of pAR1707, read throughoccurred at the Te terminator (Figure 7). There areno bands between the terminated (161 nucleotides)and the read through (502 nucleotides) transcriptsthat would be indicative of arrested elongationtranscripts. As is apparent from the data in Table1, there was a slight decrease in terminationefficiency at the Te terminator as a function ofincreasing UTP concentration during multiple-round transcription. The trends are similar at ATPconcentrations of 0.05 and 2 mM. The values ofthe termination efficiency at a UTP concentrationof 0.05 mM are comparable to the value obtainedby Reynolds et al.26 during single-round transcrip-tion at a nucleotide concentration of 0.04 mM. Thedata for multiple-round transcription indicate thatthe decrease in the amount of terminated transcriptaccumulation at elevated UTP concentrations is notdue to enhanced read through at the Te terminationsite.

Promoter clearance is modulated by UTP levels

Promoter clearance can be monitored by theappearance of short terminated transcripts as afunction of time during single-round transcription.Such studies are illustrated in Figure 8 for theDNA construct containing the T7 A1 promoterand Te terminator. At a UTP concentration of 0.05(Figure 8(a)) but not 1.0 mM (Figure 8(b)), there isa prominent band that appears to correspond topaused transcripts. The length of the paused tran-scripts is between 70 and 80 nucleotides based onDNA markers (data not shown). The size of thesepaused transcripts corresponds to the location ofthe linker DNA that Studier used to constructplasmid pARl707 (personal communications). Thelinker DNA yields nucleotides 47–68 of the

Table 1. Estimates of the termination efficiency at the T7Te terminator during multiple-round transcription as afunction of UTP concentration

[UTP] (mM) Termination efficiency (%T )

2 mM ATPa 0.05 mM ATPb

0.01 .99 99(^0.8)0.05 99 97(^0.5)0.50 98(^ l) 96(^ l.4)1.00 97(^2) 94(^0.5)

Transcript formation was monitored by the incorporation of[32P]CMP. Therefore, in the calculation of the termination effi-ciencies, the intensities of the bands were corrected for CMPcontent. The termination efficiencies were determined byusing the equation, %T ¼ ð100Þðread throughÞ=ðterminated þread throughÞ: The concentrations of UTP, GTP and [32P]CTPwere all 0.05 mM.

a Each value corresponds to the average of three independentdeterminations.

b Each value corresponds to the average of four independentdeterminations.

Figure 8. Gel electrophoretic analysis (20% (w/v) poly-acrylamide) of products formed during single-roundtranscription from the RsaI–SalI DNA fragment ofpAR1707 over a time span of five minutes in thepresence of 0.05 (a) and 1.0 (b) mM UTP at 37 8C. Eachreaction mixture contained 25.6 nM active enzymemolecules and 32 nM DNA fragment. The concentrationsof CTP, GTP and [g-32P]ATP (1500–3000 cpm/pmol)were each 50 mm. (c) Sequence and structure of predictedhairpin. This structure was determined by using version3.6 of RNAstructure (1996–2001) for Windows NT (D. H.Mathews, M. Zucker, & D. H. Turner). (d) Variations ofterminated transcripts (X), paused transcripts (W) andthe sum of terminated and paused transcripts (P) as afunction of time in the presence of 0.05 mM UTP. Allvalues in this Figure are normalized to the value of theterminated transcript at five minutes in the presence of0.05 mM UTP. The theoretical curves through the datain this Figure were obtained as indicated in Materialsand Methods. (e) Variations of the sum of terminatedand paused transcripts in the presence of 0.05 mM UTP(P) and of terminated transcripts in the presence of1.0 mM UTP (L). (f) A direct comparison of the vari-ations of terminated transcripts in the presence of0.05 mM (X) and 1.0 mM (L) UTP.


transcript that are capable of forming a hairpin(Figure 8(c)). Therefore, this site corresponds froma structural standpoint to a class I pause site.27,28

The variations in the amounts of paused and termi-nated transcripts, respectively, as a function of timeare shown in Figure 8(d) in the case of a UTPconcentration of 0.05 mM. There is a lag in theappearance of terminated transcripts and thevariation in the amount of paused transcripts goesthrough a maximum. We fitted the data by usinga model for terminated transcript formationinvolving sequential first-order reactions for pro-moter clearance and pause site escape:

The theoretical fit of the variation of paused tran-scripts as a function of time yielded unique valuesfor the rate constants with small associated errors.Analyses of three separate data sets yielded avalue of 1.9(^0.1) min21 for the rate constant forpromoter clearance and a value of 1.2(^0.2) min21

for the rate constant for pause site escape. As isapparent from the theoretical curve through thedata points in Figure 8(d), the model for promoterclearance and pause site escape involving sequen-tial first-order processes provides a good fit to theexperimental data for the variation of paused tran-scripts as a function of time. Back extrapolation ofthe experimental data corresponding to pause siteescape indicates that this pause site is 90(^20)%efficient (based on three determinations); i.e. mostof the RNA polymerase molecules pause at thissite. In the case of the variation of terminated tran-

scripts as a function of time, the theoretical fit didnot yield unique values for the rate constants forpromoter clearance and pause site escape. More-over, the associated errors were very high. As isapparent in Figure 8(d), there are two well-resolved portions of the curve for the variation ofthe paused transcripts, whereas there is simply agradual increase in the curve for the variation ofterminated transcripts. Thus, it is easier to obtainreliable and unique estimates of the respectiverate constants in the case of the paused transcripts.This is especially true if the values of the rate con-stants vary only slightly. Also shown in Figure8(d) is the variation of the sum of paused andterminated transcripts as a function of time. Thesedata obey first-order kinetics, and the value of therate constant based on the analyses of three sepa-rate data sets is 1.5(^0.2) min21.

The variation of terminated transcripts as a func-tion of time at a UTP concentration of 1.0 mM obeyfirst-order kinetics (Figure 8(e)). Also shown inFigure 8(e) is the variation of the sum of thepaused and terminated transcripts at a UTPconcentration of 0.05 mM as a function of time(broken line) from Figure 8(d). This clearly illus-trates that the clearance time at a UTP concen-tration of 0.05 mM is faster than that at a UTPconcentration of 1.0 mM. The value of the rateconstant for promoter clearance at a UTP concen-tration of 1.0 mM obtained from the analyses ofthree separate experiments is 0.9(^0.2) min21.This represents approximately a 53% decreasein the value of the rate constant for promoterclearance at a UTP concentration of 1.0 mM rela-tive to the value at 0.05 mM UTP.

Although pausing occurred at a UTP concen-tration of 0.05 mM but not 1 mM, more terminatedtranscripts accumulated during multiple-roundtranscription at a UTP concentration of 0.05 mMover a time span of 30 minutes. This is due to thefact that promoter clearance and pause site escapeat a UTP concentration of 0.05 mM are both fasterprocesses than promoter clearance at a UTPconcentration of 1.0 mM. Inspection of the dataand the theoretical fits in Figure 8(f) indicates thataccumulation of terminated transcripts at a UTPconcentration of 0.05 mM does not exceed accumu-lation of terminated transcripts at a UTP concen-tration of 1.0 mM until about two minutes afterinitiation of the reactions for this data set. Therange for all data sets is between two and fourminutes.

The above experiments were conducted at anATP concentration of 0.05 mM. Under conditionswhere the concentration of ATP was 2 mM andthat of UTP was 0.05 mM, promoter clearance wastoo rapid to allow us to obtain a value of the rateconstant for promoter clearance by using thisapproach. Therefore, we could not ascertainwhether the magnitude of the UTP effect onpromoter clearance displayed the same depen-dency on ATP concentration as that observed forterminated transcript accumulation.

Figure 9. Double reciprocal plots of 1/v versus 1/[ATP]at [a-32P]UTP concentrations of 0.05 mM (X) and 1.0 mM(B) mM. The reaction mixtures contained 2.5 nM activeRNA polymerase molecules, 3.2 nM A1 promoter andthe indicated amounts of ATP in the presence of either0.05 or 1.0 mM [a-32P]UTP (200–300 cpm/pmol). Eachpoint corresponds to the average of single time pointassays done in duplicate.


Effect of UTP on formation of firstphosphodiester bond

In the reaction between ATP and UTP as cata-lyzed by RNA polymerase from the A1 promoter,elevated concentrations of UTP inhibited the for-mation of pppApU. On the paper chromatogram,there was no radioactivity above background inregions where longer products should migrate.Therefore, the decrease in radioactivity corre-sponding to the pppApU product at elevated UTPconcentrations cannot be attributed to the con-version of pppApU to longer transcripts. To deter-mine the mechanism of UTP inhibition ofpppApU formation, we analyzed the reaction as afunction of ATP concentration in the presence of0.05 and 1.0 mM UTP, respectively. Double recipro-cal plots of the experimental data illustrating theeffect of UTP on this reaction are given in Figure9. The two lines intersect on the abscissa. Thus,UTP at a concentration of 1.0 mM is a non-competi-tive inhibitor with respect to ATP. These results areconsistent with an allosteric mechanism for UTPsuppression of pppApU synthesis. Further analysisof the data by using the equation Vmax;i ¼Vmax=ð1 þ ½UTP�=KdÞ where Vmax,i is the velocity ofthe reaction in the presence of 1 mM UTP yieldeda value of 1.6(^0.5) mM for the apparent Kd forUTP binding at the putative allosteric site. Thisvalue is nearly identical to that obtained in theexperiments on the UTP-stabilization of theopen complex against heparin disruption (i.e.Kd ¼ 1:3ð^0:4Þ mM).

The equilibria corresponding to an allostericmodel for the mechanism of action of UTP atelevated concentrations under the condition that[ATP]@ [EPo] are:

Figure 10. The reaction mixtures contained 2.5 nMactive RNA polymerase molecules, 3.2 nM A1 promoter(RsaI–SmaI fragment from pAR1707), 2 mM ATP andthe indicated amounts of [a-32P]UTP (200–300 cpm/pmol). Each point corresponds to the average of single

time point assays done in duplicate. Concentrationdependencies for the rate of pppApU formation overthe [a-32P]UTP concentration ranges of 0–0.05 mM (a)and 0–1.5 mM (b). (c) Double reciprocal plot of 1/vversus 1/[UTP] of the experimental data given in (b).The inset more clearly illustrates the fit at higher UTPconcentrations. (d) Concentration dependency for therate of pppApU formation over the ATP concentrationrange of 0–2 mM.


In this formulation, EPo–ATPA corresponds to theopen complex (EPo) in which ATP is bound atthe active site; KUTP,A and KUTP,L correspond to theapparent dissociation constants for the binding ofUTP to the active (UTPA) and allosteric (UTPL)sites, respectively; and b corresponds to the frac-tional decrease in kcat in the case of the ATP–RNApolymerase–A1 promoter open complex that hasUTP bound at the allosteric site. The velocityequation for this scheme is:

V ¼ðVmax{½UTP�=KUTP;A þ b½UTP�2=ðKUTP;AÞðKUTP;LÞ}

1 þ ½UTP�=KUTP;A þ ½UTP�=KUTP;L þ ½UTP�2=ðKUTP;AÞðKUTP;LÞ

ð1Þ

In Figure 10(a) is a plot of the variation in the rateof pppApU formation as a function of UTP concen-tration over the range of 0–0.05 mM. Analyses ofseveral data sets yielded a value of 9.3(^0.2) mMfor the apparent KUTP,A for UTP binding at theactive site and a value of 0.39(^0.01) mMpppApU/minute for Vmax. If the concentrationrange for UTP is extended up to 1.5 mM, it isapparent that UTP at elevated concentrations sup-presses the synthesis of pppApU (Figure 10(b)). Infitting the experimental data in Figure 10(b) toequation (1), the value of KUTP,L was kept constantat 1.3 mM and the other parameters were allowedto vary. This value for KUTP,L was obtained fromthe filter binding studies on the effect of UTP onthe stability of the RNA polymerase–T7 A1 pro-moter complex. The estimates for the various para-meters as determined in theoretical fits of four datasets are 0.28(^0.14) mM pppApU/minute for Vmax,8.2(^5.9) mM for KUTP,A and 0.5(^0.1) for b. As isapparent from the theoretical curve through thedata points shown in Figure 10(b), the schemegiven above involving two binding sites for UTPgives a good fit to the experimental data. Relativeto the velocity of the reaction at a UTP concen-tration of 0.05 mM, there is a 27(^5)% decrease inthe velocity at a UTP concentration of 1.0 mM.These studies were conducted at an ATP concen-tration of 2.0 mM. In comparable studies con-ducted at an ATP concentration of 0.05 mM, thevelocity of the reaction at a UTP concentration of1.0 mM was 50(^20)% less than that at a UTPconcentration of 0.05 mM (based on six determi-nations; data not shown).

In Figure 10(c) is a double reciprocal plot of thedata given in Figure 10(b). The theoretical curvethrough the data points is based on the estimatesof the various parameters (Vmax, KUTP,A, KUTP,L andb ) as determined above for this data set. At lowUTP concentrations (i.e. at high 1/[UTP] values),the double reciprocal plot is linear and it appearsthat the reaction obeys classic Michaelis–Mentenkinetics. However, as the concentration of UTPincreases, the value of 1/velocity goes through aminimum and then increases again. This increasein 1/velocity indicates substrate inhibition atelevated UTP concentrations.

To test the hypothesis that the observed variationin pppApU formation as a function of UTP concen-tration is due to product inhibition, we investi-gated the variation in pppApU formation as afunction of ATP concentration. If productinhibition is the source of the variation in pppApUproduction as a function of UTP concentration,then similar results should be obtained whetherwe vary the concentration of UTP or ATP. As isillustrated in Figure 10(d), ATP does not inhibitpppApU formation at concentrations as high as2 mM. Thus, the suppression of pppApU for-mation at elevated UTP concentrations is not dueto product inhibition. Analyses of several data setsyielded a value of 0.33(^0.01) mM pppApU/minute for Vmax, and a value of 0.12(^0.01) mMfor the apparent Km, for the interaction of ATPwith the open complex.

Discussion

Suppression of terminated transcriptaccumulation from the T7 A1 promoter atelevated UTP concentrations is due toinhibition of promoter clearance

In the presence of 0.05 mM ATP, the value of therate constant for promoter clearance at a UTP con-centration of 1.0 mM is approximately 53% lessthan that observed at a UTP concentration of0.05 mM. The magnitude of the UTP effect onpromoter clearance is sufficient to account for thesuppression of terminated transcript accumulationat elevated UTP concentrations (i.e. approximately40% suppression at 1 mM UTP relative to thevalue at 0.05 mM UTP). The difference in thesetwo values appears to be due to the pausing ofRNA polymerase molecules at a UTP concentrationof 0.05 mM. Jin and Turnbough29 observed adecrease in terminated transcript accumulation atelevated UTP concentrations in the case of a DNAfragment containing the pyr BI promoter-regulatoryregion. They attributed this phenomenon toincreased stuttering from a homopolymeric regionin the initially transcribed region of the DNAtemplate. The DNA construct containing the T7A1 promoter and Te terminator does not contain ahomopolymeric region in the initially transcribedregion that can lead to stuttering synthesis ofa string of uridine residues. Therefore, such amechanism cannot account for the decrease in theaccumulation of terminated transcripts from theA1 promoter at elevated UTP concentrations.

The magnitude of the UTP suppression of termi-nated transcript accumulation in the presenceof 2 mM ATP is less than that in the presence of0.05 mM ATP. The analysis of the mechanism ofinhibition of pppApU synthesis by UTP appearsto rule out competitive binding between ATP andUTP as a mechanism for this difference. Niermanand Chamberlin8 obtained data that suggestedthat there is an allosteric nucleotide-binding site


that may be crucial for the process of productiveinitiation. If there is indeed an ATP allostericbinding site, then ATP may partially counteractthe effect of UTP suppression of terminated tran-script accumulation through this site. Althoughthis provides a mechanism for the effect of ATP athigher concentrations, additional studies must beconducted to confirm it.

The pattern of abortive product formation as afunction of UTP concentration in the case of theA1 promoter varies from those reported in othersystems.16,18,29,30 Moreover, there is a large variationin the reported patterns of abortive productformation as a function of nucleotide con-centration.16,18,29,30 The Kms of the respective nucleo-side triphosphates, the rates of extension andrelease of each oligonucleotide in the initial tran-scribed complexes18,31,32 as well as any putativeallosteric effects probably determine each specificabortive product pattern as a function of nucleo-tide concentration. Although elevated concen-trations of UTP suppress the synthesis of both theabortive and terminated products from the A1 pro-moter, the overall UTP concentration dependenciesare different. Heisler et al.16 reported that there is asubpopulation of RNA polymerase molecules thatare slow to clear the A1 promoter sites. Sen et al.33

reported that few moribund (arrested) complexesappear to accumulate at A1 promoters. If there aretrace amounts of arrested complexes that form atA1 promoters, then the arrested complexes thatproduce the abortive products may have a differ-ent UTP concentration dependency than theproductive complexes that yield full-length termi-nated transcripts.

The rate constants for promoter clearance as wellas for pause site escape are macroscopic constantsthat describe all of the events that occur in eachprocess. Although promoter clearance from the A1promoter has been investigated previously,16,34 esti-mates of rate constants were not determined.Therefore, a direct comparison cannot be madewith the current study. The value of the rate con-stant for escape from the pause site generated bythe linker DNA (i.e. 1.2(^0.2) min21) is comparableto those for escape from naturally occurring pausesites (i.e. 0.3–4.3 min21).27,28,35 – 38 The kinetics ofpause site escape are first order in all cases. Thissuggests, as postulated previously, that the rate-determining step in pause site escape is a uni-molecular (conformational) event.4

The formation of pppApU is inhibited atelevated UTP concentrations

The reaction in which only the first two nucleo-tides in the 50 terminal sequence of the transcriptare added and the formation of the dinucleotideproduct is monitored has served as a model fortranscription initiation.39 This reaction, however,also mimics the formation of dinucleotide productduring abortive initiation. Moreover, Nierman andChamberlin8 reported abortive product formation

in the absence of productive initiation at nucleotideconcentrations below 3–4 mM in the case of the A1promoter. However, as shown here, as well asother studies,40 synthesis of abortive products stilloccurs in the case of the A1 promoter at nucleotideconcentrations greater than or equal to 10 mM. Thismay be due to trace amounts of arrested complexesthat occur at A1 promoters.33 In spite of theproblems associated with studies on the formationof pppApU from ATP and UTP, such investigationscan still provide insight into the mechanism ofaction of UTP on modulating the accumulation ofterminated transcripts from the A1 promoter.

At elevated concentrations, UTP suppresses thesynthesis of pppApU. The pattern of inhibition ofpppApU formation as a function of UTP concen-tration (in the presence of only UTP and ATP) issimilar to that seen for the suppression of termi-nated transcript accumulation as a function ofUTP concentration rather than that seen for sup-pression of abortive transcript synthesis (in thepresence of all four nucleotides) as monitored bygel electrophoresis. This suggests that the resultsobtained on the formation of pppApU from ATPand UTP more closely reflect events that occurduring productive initiation. In the presence of2 mM ATP, the velocity of pppApU formation at aUTP concentration of 1.0 mM is 27(^5)% less thanthat at a UTP concentration of 0.05 mM. Themagnitude of this change is sufficient to accountfor the suppression of terminated transcriptaccumulation during multiple-round transcriptionat elevated UTP concentrations (i.e. 23(^6)% sup-pression at 1 mM UTP concentration relative tothe value at 0.05 mM UTP) in the presence of2 mM ATP. The difference in these values is mostlikely due to pausing of RNA polymerasemolecules at a UTP concentration of 0.05 mM.These results are consistent with UTP suppressingpromoter clearance at the point of transcriptioninitiation. Mechanistic studies on the inhibition ofpppApU synthesis by UTP indicate the presenceof a low affinity allosteric binding site for UTPwhich, when occupied, decreases Vmax but doesnot alter the Km for ATP.

In previous studies on the effect of UTP concen-tration (0–80 mM) on RNA chain initiation fromthe A1 promoter, Nierman and Chamberlin41

observed no inhibition by UTP. Also, in previousstudies on the effect of UTP concentration(0–150 mM) on pppApU formation from the A1promoter, McClure et al.39 observed no inhibitionby UTP. Qualitatively these results agree with theresults reported here over the same concentrationranges. The lack of inhibition of pppApU synthesisas a function of ATP concentration (0–2.0 mM)reported by McClure et al.39 is consistent with thecurrent study.

Chamberlin & Berg42 reported both high and lowefficiency inhibition of RNA synthesis by nucleo-side triphosphates. They monitored reiterated syn-thesis of poly[r(A)] from homopolymeric regionsof denatured calf thymus DNA (single-stranded


DNA). This system and the patterns of inhibition aresignificantly different from those in the case of theDNA construct containing the T7 A1 promoter andTe terminator. Based on the many differences in thesystems, it seems unlikely that inhibition of RNAsynthesis by UTP in the case of the DNA constructused here corresponds to either the high or low effi-ciency inhibition described by Chamberlin & Berg.42

Shimamoto & Wu43,44 studied the effect of UTPconcentration on transcription initiation from apoly(dA-dT) template in which the initiatingnucleotide was UpA. The results from thesestudies led Shimamoto & Wu43,44 to propose thattwo UTP molecules bind to the enzyme–DNAcomplex during the formation of UpApU.Moreover, the second UTP acts as a positiveeffector in the process of initiation. In subsequentstudies, Shimamoto et al.45 obtained data indicatingthat UTP is not a positive effector in the initiationof transcription at the T7 A1 promoter site. This isconsistent with the results obtained here over theUTP concentration range of 0–50 mM. Shimamotoet al.45 did not investigate transcription initiationat millimolar concentrations of UTP, and thereforedid not report UTP inhibition of transcriptioninitiation. However, Shimamoto et al.45 found thatthe third nucleotide (CTP) and the fourth nucleo-tide (GTP) were potent activators of productiveinitiation at the A1 promoter and postulated thatthis activation was mediated through an allostericsite. The step that appeared to be activated bythese nucleoside triphosphates occurs after theformation of the first phosphodiester bond.

Foster et al.46 reported the allosteric regulation oftranscription elongation by nucleoside triphos-phates. In quench flow kinetic studies, theymonitored the incorporation of CMP into a stalledelongation complex as a function of CTP concen-tration. Their kinetic data were consistent with thebinding of two CTP molecules to the RNA poly-merase molecule and a mechanism involving non-essential activation by CTP. They reported valuesof 8.4 and 20 mM for the apparent dissociationconstants for the binding of CTP to the putativeallosteric binding site at approximately 23 8Cunder conditions where the active site was eitherempty or contained CTP. The relationship betweenthe CTP-activation of elongation46 and the UTP-suppression of terminated transcript accumulationreported here is unclear.

The interaction of UTP with RNA polymerase

The nucleotide-binding scheme in RNA poly-merase is fairly complex. In the case of a promoterin which the transcript is initiated by a purinenucleotide, the presence of two nucleotide-bindingsites is well established.47 The i (initiation) purinesite is template and Mg2þ-independent but ispurine nucleotide-specific. The i þ 1 (polymeri-zation) site is template and Mg2þ-dependent butdisplays no nucleotide preference. In steady-statekinetic studies, Anthony et al.48 found that the

apparent Km for the i purine nucleotide site isapproximately 0.15 mM, whereas the apparent Km

for the i þ 1 site is approximately 0.015 mM at28 8C. In equilibrium dialysis binding studiesconducted in the absence of a template but in thepresence of Mg2þ, Wu and Goldthwait49 found twobinding sites for the purine nucleotides withrespective Kds of 0.15 and 0.015 mM at 25 8C.

The results presented here add to the complexityof the nucleotide-binding scheme in RNA poly-merase. The kinetic studies establish the presenceof a low affinity UTP binding site that is distinctfrom the active site (apparent Kd ¼ 1:6ð^0:5Þ mMat 37 8C). Studies on the stabilization of the opencomplex against heparin disruption support thepresence of a low affinity UTP binding site(apparent Kd ¼ 1:3ð^0:4Þ mM at 37 8C). The fluor-escence studies on the binding of UTP to RNApolymerase in the absence of the A1 promoter alsoindicate the presence of a low affinity UTP bindingsite (0.8(^0.3) mM at 25 8C). Wu and Goldthwait49

reported a low affinity UTP binding site in equi-librium dialysis binding studies in the absence ofDNA but the presence of Mg2þ (Kd ¼ 0:5 mM at25 8C). They saw no evidence of UTP binding toRNA polymerase with an affinity correspondingto a Km (15 mM) for the binding of UTP to the poly-merization site. Moreover, they could not inducethe formation of a high affinity UTP binding siteby the addition of a purine nucleotide (GTP, GMP,dATP or dAMP). Differences in the values of Kd

for the low affinity UTP binding site may reflectdifferences in experimental conditions (tempera-ture, absence/presence of a template, presence ofother nucleotides and salt concentration) or dif-ferences inherent in the techniques that were usedto obtain estimates of this parameter. It should benoted that the equilibrium dialysis binding studiesof Wu & Goldthwait49 also indicate the presence ofa low affinity CTP binding site in the absence ofDNA but the presence of Mg2þ. It is not clearwhether CTP and UTP bind to the same lowaffinity site. Also, the relationship between theallosteric binding sites proposed in otherstudies42 – 46 and the low affinity CTP and UTPbinding sites is unclear.

The stabilization of the RNA polymerase–A1promoter complex by the binding of UTP to theputative allosteric site suggests that there is aUTP-mediated conformational change in this com-plex. Apparently, this conformational change leadsto an RNA polymerase–T7 A1 promoter complexthat is transcriptionally less active. This confor-mational change must be different from thealteration that various RNA polymerase–promotercomplexes are thought to undergo upon thebinding of the appropriate initiating nucleosidetriphosphate at the active site.

Conclusions

The data that we obtained here indicate that UTPmodulation of the accumulation of terminated


transcripts during multiple-round transcriptionfrom a DNA construct containing the T7 A1promoter and Te terminator is mediated at thepoint of transcription initiation through an allo-steric mechanism. What possible role could such amechanism serve within the cell? Under nucleo-side triphosphate concentrations within the cell,the active site of RNA polymerase is saturatedand RNA synthesis is driven as a result. Therefore,in the case of a so-called constitutive gene con-taining a strong promoter, one would expect tohave the continuous unregulated synthesis oftranscripts regardless of the amount of mRNArequired to maintain the steady-state level of agiven protein. Excess transcripts would then haveto be degraded. This would represent a waste ofenergy in the consumption of nucleoside tri-phosphates to produce mRNA transcripts that arenot needed by the cell. A mechanism wherebyRNA polymerase activity is suppressed or modu-lated at elevated nucleoside triphosphate levelswould tend to ameliorate this effect and conserveenergy. Our initial studies on the suppression ofRNA synthesis from the T7 A1 promoter atelevated UTP concentrations support such amodel for modulating RNA polymerase activity. Itshould be noted that CTP at elevated concen-trations suppresses terminated transcript accumu-lation in the case of the A1 promoter whereas GTPdoes not (our unpublished data). Thus, it appearsthat the pyrimidine nucleoside triphosphates playa role in regulating transcription from the A1promoter. Additional studies are required tofurther delineate the mechanism of action of thepyrimidine nucleotides and to obtain a more uni-fied picture of the putative role of allostericmechanisms in regulating transcription.

Experimental Procedures

Reagents and materials

[a-32P]UTP, [a-32P]CTP and [g-32P]ATP were obtainedfrom ICN. E. coli K12 cell paste (3/4 log phase, enrichedmedium) was purchased from the University ofWisconsin. Restriction enzymes were obtained fromNew England Biolabs. T4 polynucleotide kinase and calfintestinal alkaline phosphatase (CIP) were purchasedfrom Promega. All other reagents were of the highestpurity available from commercial sources. Whatmancellulose nitrate filters (25 mm and 0.45 mm pore size)and Whatman 3 MM chromatography paper werepurchased from Fisher.

Purification of RNA polymerase and isolationof holoenzyme

The method of Burgess & Jendrisak50 as modified byLowder & Johnson51 to incorporate chromatographywith a red agarose column instead of a DNA cellulosecolumn was used for the purification of RNA polymer-ase. Separation of core polymerase and holoenzyme wascarried out by chromatography on a Bio-Rex-70 (Bio-Rad) column.52 In the final step in the purification of the

holoenzyme, a modification of the method of Hageret al.53 was used involving chromatography with QSepharose FF (Pharmacia) as outlined by Johnson &Chester.52

Characterization of holoenzyme

As judged from analyses of Coomassie Blue stainedgels (sodium dodecyl sulfate–8.75% (w/v) poly-acrylamide54) by using ImageQuant, the holoenzymewas at least 90% saturated with sigma. The number ofactive enzyme molecules present in the samples was esti-mated by using the procedure of Oen et al.55 as modifiedby Solaiman & Wu.56 The protocol is presented in detailby Johnson & Chester.52 In the studies conducted herein,the percentage of active enzyme molecules was approxi-mately 40%.

Isolation of plasmids and DNA fragments

Dr W. Studier (Biology Department, BrookhavenNational Laboratory, Upton, NY) generously providedplasmids pAR1707 and pAR1435. Plasmid pAR1707 con-tains the A1 promoter site as well as the early termi-nation site Te from bacteriophage T7, whereas plasmidpAR1435 contains just the T7 A1 promoter cloned intothe BamH1 site of pBR322. Bacteria (DH5a competentcells containing either pAR1707 or pAR1435) weregrown in LB medium (1% (w/v) Bacto-tryptone, 0.5%(w/v) Bacto yeast extract, 1% (w/v) NaCl) containing40 mg/ml ampicillin according to standard protocol.57

Plasmids were isolated by using Qiagen columns asdescribed in the protocol provided by the manufacturer.After digestion of the plasmids with the appropriaterestriction enzymes, the fragments were separated onnative 5% polyacrylamide gels. The electrophoresisbuffer was TBE (90 mM Tris, 90 mM boric acid, 2.5 mMEDTA, pH 8.3). The DNA fragment of interest was thenisolated by electroelution from the gel. This was fol-lowed by volume reduction with sec-butanol, phenolextraction and then precipitation with ethanol. The pre-cipitated fragment was then resuspended in buffer(10 mM Hepes, 50 mM KCl, 10 mM MgCl2, 0.1 mMEDTA, 10% (v/v) glycerol, 0.1 mM DTT at pH 8.0).Treatment of pAR1707 with RsaI and SmaI yielded a 529base-pair fragment containing the A1 promoter; treat-ment of pAR1707 with RsaI and SalI yielded a 963 base-pair fragment containing the A1 promoter along withthe Te terminator; and treatment of pAR1435 with RsaIand PvuII yielded a 2030 base-pair fragment containingthe A1 promoter.

Radioactive labeling of the DNA fragment forheparin displacement studies

The SmaI–RsaI fragment from pARl707 was dephos-phorylated by using CIP according to standardprotocol.57 Labeling with 32P was performed by mixingthe dephosphorylated DNA fragment with [g-32P]ATPin the presence of T4 polynucleotide kinase according tostandard protocol.57 Unreacted [g-32P]ATP was removedby electrophoresis on a native 5% polyacrylamide gel.The protocol for electrophoresis and isolation of thelabeled DNA fragment was the same as that givenabove for the isolation of DNA fragments from restric-tion digests.


Transcript analyses

All reactions throughout this study were conducted inHepes buffer (10 mM Hepes, 50 mM KCl, 10 mM MgCl2,0.1 mM DTT at pH 8.0) at 37 8C. The purchased nucleo-tide samples were in the salt form. Therefore, whennecessary, the salt concentration of each reaction mixturewas adjusted to account for the salt added due to therespective nucleotides. All solutions were preincubatedat 37 8C for ten minutes prior to initiation of the reaction.In the case of multiple-round transcription, the reactionvolume was 20 ml and each reaction was terminated bythe addition of 10 ml of stop reaction mixture (0.2 MEDTA, 0.01% (w/v) bromophenol blue, 50% glycerol).For monitoring the recycling phase as a function of timeduring multiple-round transcription, 10 ml aliquots wereremoved from the reaction mixture (80 ml) at the appro-priate times and the reaction was stopped by theaddition of 10 ml of stop reaction mixture. In the case ofpromoter clearance, the reaction was limited to a singleround by the addition of heparin (final concentration of40 mg/ml) along with the nucleotides. Aliquots of 10 mlwere removed from the reaction mixture (80 m1) at theappropriate times and mixed with 10 ml of stop reactionmixture. After stopping each reaction, the samples wereheated in boiling water for one minute and then cooledon ice. The samples (10 ml) were applied to either an8 M urea 20% or 8% (w/v) polyacrylamide gel and sub-jected to electrophoresis in TBE buffer. The gels weredried and then exposed to X-ray film (Fuji RX) at eitherroom temperature or at 270 8C for varying lengths oftime. The autoradiograms were analyzed by usingImageQuant (Molecular Dynamics).

Heparin displacement assay

The stability of the open complex between RNA poly-merase and the A1 promoter (32P-end labeled RsaI–SmaIfragment of pARl707) in the absence and presenceof UTP was determined by measuring retention ofcomplexes on nitrocellulose filters after challenge withheparin (50 mg/ml) in Hepes buffer at 37 8C. After apply-ing the respective samples to the filters, they wereimmediately filtered under vacuum. Each filter wasthen washed five times with 1 ml of Hepes buffer. Thefilters were dried in an oven at 50 8C for one hour andthen subjected to Cerenkov counting. Background reten-tion of the end-labeled DNA fragment was determinedby filtering a reaction mixture containing everythingexcept for RNA polymerase. In all cases, the backgroundretention was less than 3%. For determination of the dis-ruption of the open complex as a function of time, 50 mlaliquots were removed at appropriate times from thereaction mixture (350 ml) after the addition of heparin.For determination of the UTP concentration dependencyfor stabilization of the RNA polymerase–T7 A1promoter complex against heparin disruption, 45 ml ofeach reaction mixture (50 ml) was removed after 15minutes and analyzed as outlined above.

Transcription elongation assay

The A20 elongation complex was generated in theabsence of a labeled radioactive nucleotide by incubatingApU, GTP, ATP and CTP (all at 0.05 mM) with a solutioncontaining RNA polymerase (18.5 nM active enzymemolecules) and T7 A1 promoter (24 nM RsaI–PvuIIDNA fragment from pAR1435) complexes in Hepesbuffer. The nucleotide reaction mixture also contained

heparin (60 mg/ml) to ensure that no initiation occurredafter formation of the A20 complexes. After a five minuteincubation, nucleotide solutions containing ATP(0.05 mM), GTP (0.05 mM), [a-32P]CTP (0.05 mM at aspecific activity of 700–1000 cpm/pmol) and the appro-priate amounts of UTP were added and 25 ml aliquotswere removed from the reaction mixture (350 ml) atappropriate times for analyses. RNA polymerase finalconcentration was 12 nM in active enzyme molecules,the T7 A1 promoter final concentration was 16 nM andheparin final concentration was 40 mg/ml. The aliquotswere treated as described by Kingston et al.58

Fluorescence studies

All fluorescence studies were conducted by using aSpex Model 1681 spectrofluorometer that was interfacedto a Spex DM3000 computer. Measurements were madein the ratio mode by using front-face illumination.59

Excitation and emission bandwidths were 1.8 and4.5 nm, respectively. With front-face illumination, alinear dependence of fluorescence intensity on proteinconcentration was observed up to at least 2000 nM.Therefore, no corrections were required for inner filtereffects. For fluorescence emission spectra, data wererecorded every 1 nm with an integration time of onesecond. The excitation wavelength was 280 nM. Eachspectrum was corrected for blank background emissionand wavelength-dependent effects. All studies wereconducted at 25 8C.

In the fluorometric titration of RNA polymerase byUTP, 11 measurements were made at 340 and 500 nm,respectively, at each UTP concentration. An integrationtime of two seconds was used in recording each datapoint. These measurements were then averaged and thedifferences between the measurements at 340 and500 nm were determined and corrected for dilution.

Analysis of promoter clearance data

The model that we used to analyze the data obtainedat a UTP concentration of 0.05 mM corresponds tosequential pseudo first-order processes for promoterclearance and pause site escape. Harcourt and Esson60

were the first to report the integration of the differentialequations for this type of series first-order reactions. Byusing a variation of these equations as presented byFrost and Pearson,61 we fitted the experimental data inorder to obtain estimates for k1 and k2. In the case of thevariation of the paused transcript as a function of time,the data were fitted to the equation:

g ¼ 1 þ ½1=ð1 2 k2=k1Þ�½ðk2=k1Þexpð2k1tÞ2 expð2k2tÞ�

where g is the normalized value for the amount ofpaused transcripts present and t corresponds to time. Inthe case of the variation of terminated transcripts as afunction of time, the data were fitted to the equation:

b ¼ ½1=ðk2=k1Þ2 1�½expð2k1tÞ2 expð2k2tÞ�

where b is the normalized value for the amount of theterminated transcripts. In the analysis of the data for thesum of the terminated and paused transcripts at0.05 mM UTP or the data for the terminated transcriptsat 1.0 mM UTP, the equation:

a ¼ 1 2 expð2ktÞ

was used where a is the corresponding normalizedvalue. All fits of experimental data were done by using


Sigmaplot 4.0 for Windows and the appropriateequation.

Steady state kinetic analysis oftranscription initiation

The steady state kinetics of product formation(pppApU) between UTP and ATP were performed byusing the procedure of McClure.62 Reaction mixtures(30 ml) contained in Hepes buffer RNA polymerase(2.5 nM active enzyme molecules), A1 promoter(3.2 nM), and the appropriate amounts of ATP and[a-32P]UTP at a specific radioactivity of 360 cpm/pmol.Prior to the initiation of the reaction, the solutions werepreincubated for ten minutes at 37 8C. Each reaction wasterminated at the appropriate time by the addition of10 ml of 0.2 M EDTA and then placed on ice. The sampleswere spotted on Whatman 3 MM paper in 5–6 mlaliquots with interim drying and resolved withascending chromatography in WASP (water/saturated(NH4)SO4 (pH 8.0)/2-propanol, 18:80:2 (by volume) con-taining 5 mM EDTA). The chromatograms were dried atroom temperature, cut into strips and analyzed for radio-activity by Cerenkov counting. The Rf value of pppApUwas approximately 0.44. In studies in which the concen-tration of [a-32P]UTP was varied over the concentrationrange of 0–1.5 mM, a standard curve based on the back-ground radioactivity due to unincorporated [a-32P]UTPthat migrated to the area corresponding to a Rf value of0.44 was used to obtain the corrected value for pppApUformation.

Acknowledgments

This research was supported, in part, by FacultyResearch Grant 2-65503 from the Brody School ofMedicine at East Carolina University.

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Edited by D. Draper

(Received 18 September 2001; received in revised form 31 January 2002; accepted 2 February 2002)


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