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In Vitro Comparison of Initiation Properties of Bacteriophage X Wild-Type PR and X3 Mutant Promoters (RNA Polymerase Mechanism/Abortive Initiation) DIANE K

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In Vitro Comparison of Initiation Properties of Bacteriophage X Wild-Type PR and X3 Mutant Promoters (RNA Polymerase Mechanism/Abortive Initiation) DIANE K Proc. Natl. Acad. Sci. USA Vol. 77, No. 11, pp. 6381-6385, November 1980 Biochemistry In vitro comparison of initiation properties of bacteriophage X wild-type PR and x3 mutant promoters (RNA polymerase mechanism/abortive initiation) DIANE K. HAWLEY AND WILLIAM R. MCCLURE Department of Biochemistry and Molecular Biology, Conant Laboratory, Harvard University, Cambridge, Massachusetts 02138 Communicated by Mark Ptashne, July 28,1980 ABSTRACT The in vitro initiation properties of the PR Our analysis is based on a simple two-step model of pro- promoter of bacteriophage X and of a PR mutant, x3, were moter-polymerase interaction first proposed by Zillig and his compared. Using the abortive initiation reaction, we measured coworkers (4). According to this model, RNA polymerase first the lags in the approach to a final steady-state rate when dinu- binds to the DNA in a transcriptionally inactive "closed" cleotide synthesis was initiated with RNA polymerase. These unwinds the DNA to form the lags corresponded to the average times required for the forma- complex (RPc) and subsequently tion of transcriptionally active open complexes. By measuring "open" complex (RPO) which then binds the nucleoside tri- the lags at different RNA polymerase concentrations, we could phosphates and initiates transcription (5). Formation of the open separate open complex formation into two steps, based on a complex can be described as follows: simple model in which the initial bimolecular association of free ks k2 promoter and polymerase in a closed complex is followed by an R + Pa SRPco- RPo. [1] isomerization to the open complex. The contribution of each k-1 k-2 step to the overall rate of open complex formation was quanti- tated for both promoters. We found that the x3 mutation, which In this study, we used the abortive initiation assay to monitor is located in the -35 region of PR, resulted in a decrease in the the lags in the formation of open complexes on the two pro- association constant for the initial binding to the closed complex moters when the reaction was initiated with RNA polymerase. to 5% of its wild-type value and a decrease in the rate of the The average time necessary for open complex formation, 'robs, isomerization to 20%. The lifetimes and abortive initiation was measured at different RNA polymerase concentrations. characteristics of the mutant and wild-type promoters were similar. We concluded that the main effect of the x3 mutation These data were analyzed in a way that allowed the equilibrium was to increase the average time of open complex formation and constant for the initial binding (KI = k1/k_1) to be quantitated that the functional properties of the open complexes did not separately from the rate of open complex formation (k2). This differ significantly between the two promoters. latter process is referred to as an isomerization because we do not know whether we have measured the rate of DNA melting Promoters are regions of DNA to which RNA polymerase binds or another rate-determining conformational change. The results to begin RNA synthesis. The structural features of promoters of our study indicate that the main effect of the x3 mutation and the details of the polymerase-DNA interaction are subjects in vitro is a decrease in K, to 5% of the wild-type value and a of great interest because the initiation of transcription is an decrease in k2 to 20%. important control point in the regulation of gene expression. Promoters recognized by Escherichia coli RNA polymerase MATERIALS AND METHODS contain two regions of sequence homology: (i) the Pribnow box, a six-base-pair sequence centered at -10 with respect to the DNA Templates and Enzyme. The source of X PR was the startpoint of transcription, and (ii) another six-base-pair se- 890-base-pair DNA fragment isolated after Hae III digestion quence located around -35. RNA polymerase interacts with of purified bacteriophage X DNA. The X x3 promoter was bases in the Pribnow box and -35 region, as determined by obtained by Hae III digestion of a pBR313-derived plasmid, chemical probe experiments. The importance of these se- pRB70, constructed by Roger Brent (Harvard University). This quences to in ivo polymerase recognition is demonstrated by plasmid contained an insertion of XcI857x3 DNA spanning the the occurrence of most promoter mutations within these two region from the EcoRI site in the 0 gene to the Bam I site to regions (1, 2). the left of the N gene. The x3 mutation abolishes HindII cutting The frequency of transcription initiation at some promoters within the promoter (3); this characteristic was confirmed for is regulated by accessory proteins, such as repressors and acti- the A Hae III 890 fragment obtained from this plasmid (un- vators. Other promoters appear not to require additional protein published data). DNA fragments resulting from Hae III di- factors; indeed, mutations that affect transcription from a gestion of phage or plasmid DNA were separated on a 3.5% promoter both in vio and in vitro demonstrate that the DNA polyacrylamide gel and extracted from the gel with phenol (6). sequence alone can directly determine the frequency of initi- The DNA was separated from UV-absorbing impurities by ation on a promoter. One such mutant, x3, is a base-pair change DEAE-Sephadex (A-25) chromatography as described (7) or in the -35 region of PR, the major rightward promoter of by spermine precipitation (unpublished data). DNA concen- bacteriophage X (3). We studied the initiation properties of x3 trations were calculated on the basis of an absorption coefficient and PR to determine what steps in the RNA polymerase- pro- at 260 nm of 6.5 mM-1 cm-1 DNA phosphorus and are ex- moter interaction have been affected by this base pair pressed as molar fragment. E. coli RNA polymerase was isolated by the procedure of Burgess and Jendrisak (8). Holoenzyme was change. separated from core according to Lowe et al. (9). Enzyme = and a molecular The publication costs of this article were defrayed in part by page concentrations are based on E" nm 6.2 charge payment. This article must therefore be hereby marked "ad- weight of 4.9 X 105 (10). The enzyme was 50-60% active in vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: polyfd(A-T)l, poly[d(A-T)]-poly[d(A-T)]. 6381 Downloaded by guest on September 29, 2021 60382 Biochemistry: Hawley and McClure Proc. Natl. Acad. Sci. USA 77 (1980) promoter binding and initiation as judged by titrations of promoter-containing DNA fragments according to Cech and McClure (11). Determination of Tobs. Details of the abortive initiation reaction have been described (7, 12). For each determination of Tobr, a control nucleotide-initiated reaction was run in parallel with a reaction initiated by addition of RNA polymerase. Both 10 reaction mixtures had a final volume of 250 Ml. For the nucle- x otide-initiated reaction, the DNA in 150 ul of reaction buffer was preincubated at 370C for 3 min before addition of 50 pl of RNA polymerase in reaction buffer. The DNA and RNA 5- polymerase were preincubated together for 14 min when the template was PR or 33.5 min when the template was x3. After this preincubation, AMP (or CpA) and UTP in 50 AI of reaction buffer were added to initiate the reaction. For the polymer- 0 10 20 30 40 ase-initiated reaction, the AMP and UTP were preincubated Time, min with the DNA in 200 Atl of reaction buffer for 5 min before addition of 50 of RNA polymerase at zero time. Aliquots of FIG. 1. Lag in the approach to a final steady-state rate ofabortive Al initiation from the x3 promoter. The radioactivity incorporated into each reaction mixture were spotted onto Whatman 3MM paper pApU is plotted versus reaction time. The standard assay conditions and developed with ascending chromatography in H20/satu- were: 40 mM Tris-HCl, pH 8; 10 mM MgCl2; 120 mM KCl; 1 mM di- rated ammonium sulfate/isopropanol, 18:80:2 (vol/vol), as thiothreitol; 100 jig of bovine serum albumin per ml; 2 mM AMP; 0.05 described (7). mM UTP ([a-32PJUTP was added to a specific activity of 500-600 The rate of the control nucleotide-initiated reaction was cpm/pmol); and 1 nM x3-containing X Hae 890 fragment in 250 ,u determined by a linear least squares analysis- rT was calculated total volume. The RNA polymerase concentration was 133 nM. The steady-state rate of the control nucleotide-initiated reaction (0) was from the polymerase-initiated reaction by extrapolating a least 22 pApU promoter'1 min-'. The curve corresponding to the poly- squares line calculated for data points corresponding to intervals merase-initiated reaction (o) was calculated from a rob. of 10.3 min greater than 4 times the estimated robs to the time axis. These and a final steady state rate of 20 pApU promoter-1 min-1. calculated values for robs and the final steady-state rates were then used as initial estimates of the values determining a single average time required for the free enzyme and promoter to exponential curve. A final value for Tob was obtained from the combine and to isomerize into the open complex. curve best fitting the experimental data. The nucleotide components of the abortive initiation reaction Determination of Open Complex Lifetimes. Because the were varied to determine whether the observed lags were af- lifetimes of the RNA polymerase-promoter complexes were fected by some characteristic of the steady-state reaction. The very long, we chose to measure the initial rate of decay of the robs values determined by monitoring pApU, pppApU, or complex by heparin and poly[d(A-T)]-poly[d(A-T)] (abbre- CpApU synthesis were the same within experimental error, viated herein as poly[d(A-T)]) challenge techniques and to although the steady-state rate of UTP incorporation into the calculate koff from a semilogarithmic plot of fraction of open three products was different in each case.
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