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Mutations Affecting Two Different Steps in Transcription Initiation at the Phage a PRM Promoter (RNA Polymerase/A Promoters) MING-CHE SHIH and GARY N

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Mutations Affecting Two Different Steps in Transcription Initiation at the Phage a PRM Promoter (RNA Polymerase/A Promoters) MING-CHE SHIH and GARY N Proc. NatL Acad. Sci. USA Vol. 80, pp. 496-500, January 1983 Genetics Mutations affecting two different steps in transcription initiation at the phage A PRM promoter (RNA polymerase/A promoters) MING-CHE SHIH AND GARY N. GusSIN* Genetics Ph. D. Program and Department of Zoology, University of Iowa, Iowa City, Iowa 52242 Communicated by A. D. Kaiser, October 4, 1982 ABSTRACT The abortive initiation assay [McClure, W. R. that the consensus sequence found at -35 in most Escherichia (1980) Proc. NatL Acad. Sci USA 77, 5634-5638] was used to study coli promoters (11, 12) provides information necessary for the the effects of mutations on the activity of the PRM promoter of initial binding of RNA polymerase to DNA and the consensus phage A in vitro. The transcription initiation properties of four sequence at -10 is required for DNA strand separation and the mutant promoters were compared with those ofwild-type PRM in formation of open complexes. the presence or absence of repressor (which activates PRM). Two kinetic parameters were measured: k2, the rate constant for the transition between closed and open complexes, and KB, the equi- MATERIALS AND METHODS librium constant for the initial binding ofRNApolymerase to DNA RNA Polymerase, Repressor, and DNA. RNA polymerase (formation of closed complexes). The primary effect of repressor holoenzyme was purified the method of and on wild-type initiation was stimulation of the isomerization reac- by Burgess Jen- tion: k2 increased about 7-fold. Both in the presence and in the drisak (13). Enzyme preparations were 90-95%fc pure as judged absence of repressor, prmU31 and prmEl04 (changes at nucleo- by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, tides -33 and -38, respectively) reduced KB significantly without contained 0.7-0.9 mol of o- subunit per mol ofpolymerase, and affecting k2, indicating that these mutations affect polymerase were 24-40% active. The activity of purified enzyme was de- binding but not the formation of open complexes. In contrast, termined by the method of Cech and McClure (14) or by as- prmE37 (a change at nucleotide -14) reduced k2 significantly saying the amount of radiolabeled DNA (containing a single without affecting KB. A fourth mutation, prmE93 (at nucleotide promoter) retained by nitrocellulose filters at saturating poly- -39), is phenotypically Prm primarily because it causes a defect, merase concentrations. Purified A repressor was a gift from C. in the OR2 operator site and, therefore, the mutant promoter is Pabo. The DNA template was an 889-base-pair Hae III restric- unable to respond normally to repressor. These results are con- tion fragment isolated from wild-type or mutant phage accord- sistent with the idea that the two regions of Escherichia coli pro- ing to described procedures (10). moters in which consensus sequences have been identified, the Abortive Initiation. The abortive initiation assay has been regions at nucleotides .-35 and -10, may provide information for described by McClure (6). Standard incubation mixtures in- two discrete steps in transcription initiation. cluded 0.04 M Tris'HCl, pH 8.0; 0.1 M KCl; 0.01 M MgCl2; 1 mM dithiothreitol; 0.05 mM UpA [uridylyl(3'-5')adenosine]; According to current models, there are two main steps in tran- 0.05 mM [3H]UTP (1 Ci/mmol; 1 Ci = 3.7 x 10'° becquerels); scription initiation prior to the polymerization ofribonucleoside 1 or 2 nM DNA fragment (as indicated); and indicated concen- triphosphates (NTPs) into RNA chains: (i) binding ofRNA poly- trations ofactive RNA polymerase. To measure Tobs (the average merase to DNA to form closed complexes,- followed by (ii) the time required for open complex formation), DNA, enzyme, and transition (isomerization) ofclosedcomplexes to open complexes substrates were incubated at 370C. At various times, 20-1.l ali- (1, 2). The second step, which appears to involve DNA strand quots were removed and assayed chromatographically for separation (2-4), is followed by very rapid RNA chain initiation UpApU (6). For repressor activation reactions, the DNA frag- in the presence of NTPs (5, 6). A method recently developed ment was incubated with repressor at 370C for 10 min prior to by McClure (6) permits the determination of KB, the, equilib- the addition of polymerase and NTPs. rium constant for the binding reaction, and k2, the forward rate Calculation ofTobs The steady-state rate of UpApU synthesis constant for the isomerization reaction, in vitro. We have used was estimated from the slope of the curve in the period cor- this method to study the effects of mutations on the PRM pro- responding to 3-5 times Tobsv Startingwith estimated values of moter of bacteriophage A. This promoter directs the synthesis the slope and Tobs, a least-square computer analysis was used of the phage-specific repressor (cI gene product), which is an to find the best fit to the equation autogenous positive regulator of transcription initiation at PRM N = Vt - Vrb, (1 -e /Tobs), (7, 8). [1] We have determined KB and k2 for wild-type PRM and four in which N = total UpApU concentration, V = steady-state rate mutant promoters in both the presence and absence ofrepres- of UpApU synthesis, and t = time of incubation (6). sor. The Prm- phenotypes and nucleotide sequence changes Fixed-Time Assays. For some experiments with prmE93 associated with the mutations have been described previously (Fig. 5), the "fixed time assay" (15) was used. Enzyme and DNA (9, 10). As expected, all four mutations cause defects in tran- were mixed at time zero in the absence ofsubstrates. At various scription initiation in vitro. In addition, the properties of the times thereafter, aliquots were removed and incubated with wild-type and mutant promoters are consistent with the idea substrates for 3 min. The reaction mixtures were then assayed chromatographically for UpApU. Incubation conditions were The publication costs ofthis article were defrayed in part by page charge the same as for abortive initiation assays described above except payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom all correspondence should be addressed. 496 Downloaded by guest on September 26, 2021 Genetics: Shih and Gussin Proc. Natl. Acad. Sci. USA 80 (1983) 497 that the specific activity of [3H]UTP was increased to 2.5 Ci/ mmol. Substrates. UpA was purchased from Collaborative Research (Waltham, MA); [3H]UTP (10 Ci/mmol) was purchased from Amersham-Searle (Chicago, IL). RESULTS Abortive Initiation at PRM. In a typical abortive initiation reaction, only the substrates corresponding to the first two nu- cleotides in aparticular transcript are supplied. The initial dinu- cleotide is synthesized repeatedly by each RNA polymerase- promoter open complex without dissociation of the enzyme from the promoter (16). Thus, at any time, the rate of dinu- cleotide synthesis is a measure of the number of open com- plexes present. For PRM, the usual substrates for the abortive initiation reaction are ATP and UTP (Fig. 1). However, the DNA template used in these experiments, the Hae III 889-base- pair restriction fragment ofA, contains both PRM and PR. There- 20 40 60 80 100 fore, to avoid confusion with PR, which also initiates transcrip- Time, min tion with pppApU, the dinucleotide UpA is used as a substrate FIG. 2. Determination of ib.. Theprm' 889-base-pairHae Im re- in place ofATP. Hawley and McClure (15) have shown that this striction fragment (2 nM) was incubated with RNA polymerase (60 substitution limits initiation to PRM because UpA corresponds nM) in the presence of substrates. Aliquots were withdrawn at indi- uniquely to the PRM sequence at -1/+1 (Fig. 1). cated times to assay for the formation of UpApU. In one case (e), en- The results ofa typical abortive initiation assay are illustrated zyme was incubated with DNA for 60 min prior to addition of sub- in Fig. 2. In the control reaction, RNA polymerase (60 nM) was strates; in the othercase (o), enzyme, DNA, andsubstrates were added incubated with the DNA template for 60 min at 37C to allow together at time zero. Extrapolation of the slope of the second exper- complete formation of open complexes prior to the addition of iment to the abscissa (----) yields rob. substrates. The addition of UpA and UTP resulted in immediate synthesis of UpApU at the steady-state rate. In a parallel re- spectively; KB is the equilibrium constant for the initial binding action, RNA polymerase, DNA, and substrates were added si- reaction; and k2 and k2 are the association and dissociation rate multaneously at time zero. In this case, there is a noticeable lag constants for isomerization. Under the conditions used in these period before the synthesis of UpApU reaches the steady-state experiments, Tob, is related to the initial RNA polymerase con- rate; the lag period is the average time necessary for open com- centration, [RNAP], by the equation (6) plex formation. Theoretically (6), the limiting rates for the two 1 I reactions should be the same; this is indeed the case for the 'obs = - + [2] experiment illustrated in Fig. 2. Extrapolation of the time k2 k2KB[RNAP] course for the second reaction to the abscissa yields Tobs (the lag Therefore, a plot of Tobs versus 1/[RNAP] should be linear with time), which was about 20 min at an RNA polymerase concen- intercept equal to 1/k2 and a slope equal to 1/k2KB, in which tration of 60 nM. 1/k2 = -, the average time necessary for the isomerization re- Determination of x, k2, and KB. Transcription initiation can action. be diagrammed as follows (1, 2, 6): Plots ofTob, versus 1/[RNAP] for wild-type and three mutant KB k2 +NTPs promoters incubated with RNA polymerase in the absence of E + D_ E-DC _ E-Do- RNA synthesis, repressor are shown in Fig.
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