Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3134-3138, August 1974 Interaction of the Operator of the Tryptophan Operon with Repressor (repressor-operator interactions/trp repressor/in vitro transcription/Escherichia coli) JOHN K. ROSE AND CHARLES YANOFSKY Department of Biological Sciences, Stanford University, Stanford, California 94305 Contributed by Charles Yanofsky, April 23, 1974 ABSTRACT Transcription studies in vitro on repres- where stated otherwise, the ratio of polymerase to DNA was sion ofthe tryptophan operon of Escherichia coli show that maintained at 1:1 (w/w). Each reaction mixture also con- partially purified trp repressor binds specifically to DNA containing the trp operator with a repressor-operator tained 6 M1A of buffer IV (1) with the indicated amounts of dissociation constant of about 0.2 nM in 0.12 M salt at partially purified repressor. Buffer IV added final concentra- 370, a value consistent with the extent of trp operon tions of 2 mM Tris-OAc (pH 8.0), 12 mM KOAc, 2.5 mM regulation in vivo. The half-life of the trp repressor-trp Mg(OAc)2, 0.14 mM dithiothreitol, and 1% glycerol. RNA operator complex is less than 2 min in vitro in 0.12 M salt. synthesis, RNA purification, and DNA template preparation Partially purified trp aporepressor (1) represses transcription were as described (2). The initial steps of aporepressor puri- in vitro of the tryptophan (trp) operon of Escherichia coli in the fication were as described (1), and then the aporepressor was presence of itryptophan or appropriate tryptophan analogues taken through an additional DEAE-cellulose column step (2-4). Neither tryptophanyl-tRNA synthetase (3, 5) nor which gave 10-fold further purification (5). The final prepara- tryptophanyl-tRNA (5-7) appears to participate in repression. tion of aporepressor was estimated to be 550-fold purified over Repression in vitro involves interaction between the trp that in crude extracts. This preparation contains about 1 nM repressor and the trp operator, since an operator constitutive aporepressor at a protein concentration of 5.2 sg/ml (Fig. 2). mutation eliminates repression (2). On the basis of these find- Assuming a molecular weight of 58,000 (1, 5), the active apo- ings it may be tentatively concluded that the protein product repressor would constitute about 1.0% of the protein in the of trpR, the aporepressor, when complexed with the corepres- preparation. sor, L-tryptophan, acts at the trp operator to present transcrip- RESULTS of tion the operon. The template DNA used in most of the transcription experi- Attempts to assay trp repressor-trp operator interaction from a specialized assays (8) have been ments in vitro described here was isolated by conventional DNA-protein binding transducing phage, 080 trpO+ AtrpEDl02 trpCBl, which unsuccessful. Therefore, we have used the in vitro transcrip- of trpA, and this interaction. carries the trp operator, trpC, trpB, and a portion tion system to study has a particular deletion of trpE and D. This deletion results MATERIALS AND METHODS in relatively high rates of trpCB mRNA synthesis in vivo and Bacteriophage Isolation. The transducing phages 480 trpO+ in vitro (10, 2). trpCB mRNA is detected by hybridization of AtrpED102 trpCBl and XtrpCBl have been described (2). synthesized labeled mRNA to the separated strands of the carries and the operator proximal DNA of XtrpCB1. In agreement with previous results (2), we Phage 080 trpO+ trpE7 trpE from the portion of trpD and was isolated after induction of an E. coli find that approximately 8% of the RNA transcribed the deletion tonB trpAD32. This dele- 4)80 trpO+ AtrpEDl02 trpCBl template by purified RNA poly- 480h- lysogen carrying correct or c strand of XtrpCB1 DNA, tion removes the operator distal genes trpA, trpB, and trpC, merase hybridizes to the carries the trp and less than 1% hybridizes to the incorrect or i strand of the and about 80% of trpD. Phage 480 trpO+ trpE7 to X as shown formation anal- latter DNA. A blank value of 0.3-0.4% hybridization operator and promoter, by enzyme In ex- similar to those described by DNA has been subtracted from these percentages. all yses in infection experiments in excess Franklin (9). The gene order in the operon is trp operator (0)- periments described below, tryptophan was present at a concentration of 0.3 mM, 30-fold above that required for E-D-C-B-A. half-maximal repression (2). Reaction Conditions In Vitro and Repressor Preparation. In vitro reactions (2) were for 15 min at 37°. The final volume Determination of the dissociation constant of each reaction mixture was 30 /l, containing 0.3 mM L- Fig. 1 shows the percent repression of correct strand trpCB tryptophan, 12 mM Tris- HC1 (pH 7.9), 1.3 mM MgCI2, 97 mRNA synthesis at various repressor concentrations. Each mM KCl, 0.13 mM EDTA, 0.1 mnM dithiothreitol, 0.09 mM curve presents results obtained at a different template DNA ATP, CTP, and GTP, 0.017 mM [3H]UTP (10 Ci/mmole), concentration with the ratio of polymerase to DNA held con- 10 /Ag/ml (0.33 nM) to 150 ,g/ml (5 nM) of DNA and 10 /ug/ stant. Note that as the DNA concentration is increased (at ml to 153 ,ug/ml of RNA polymerase holoenzyme (EC 2.7.7.6; repressor preparation levels of less than 8 ,ug/ml), there is a nucleosidetriphosphate: RNA nucleotidyltransferase). Except decrease in percent repression of trp operon transcription. Note also that at low DNA concentrations a change in DNA Abbreviations: trp, tryptophan gene or operon; c strand and i concentration has only a relatively small effect on repression, strand, correct and incorrect strand, respectively, of XtrpCB1 while at higher DNA concentrations (>1 nM) the effect is DNA. large. For example, a repressor concentration that is just 3134 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) trp Operon Regulation 3135 sufficient to give 50% repression with 0.33 nM DNA shows only a 1.3-fold reduction in percent repression (to 38%) when the DNA concentration is increased 3-fold to 1 nM. A much larger (3.8-fold) reduction in repression (to 10%) is seen when the DNA concentration is increased another 5-fold to 5 nM. These results suggest that at the low DNA concentrations .2a tested there are significant levels of free repressor and free 0co,ID operator. Thus, if we assume that percent repression indicates 4e the percent of operators present that are bound to repressor, 10% repression of 5 nM DNA would be equivalent to a repres- sor concentration of at least 0.5 nM. This same repressor con- centration, however, gives only 50% repression of 0.33 nM DNA, although the repressor concentration must be higher than the DNA concentration. Therefore, in this case, we con- clude that more than half of the repressor is not bound to trp Partially Purified Repressor (mg/ml) operators. Similar reasoning leads to the conclusion that the FIG. 1. Repression of trp operon transcription as a function of of the molecules are bound to DNA and repressor concentrations. Reaction conditions in vitro majority trp repressor present and partially purified repressor were as described in Materials and trp operators at the higher DNA concentrations used in these Methods. The indicated concentrations of partially purified experiments. The above observations suggest an equilibrium repressor and 0.3 mM L-tryptophan were preincubated with between trp repressor, trp operator, and the trp repressor-trp template DNA for 2 min at 00 in the standard reaction mixture. operator complex, with the value for the dissociation constant RNA polymerase was added and incubation was continued for of the complex near the concentrations of repressor and opera- anadditional 2min at 00. Reactions were initiated by addition tor. of nucleoside triphosphates and were run for 15 min at 37°. We shall show below that these data are consistent with a Template (080 trpO+ AtrpED102 trpCBl DNA) concentrations simple equilibrium situation described as follows:* were: A, 0.33 nM; A, 0.5 nM; 0, 1 nM; *, 2 nM; 0, 2.5 nM; X, 5 nM. The RNA polymerase:DNA ratio was constant at each R + 0 2 RO [1] DNA concentration. Total 3H cpm in RNA ranged from 18,651 (0.33 nM DNA) to 285,033 (5 nM DNA). At each DNA con- The dissociation constant at equilibrium for this reaction is centration, trp CB c strand mRNA (measured by hybridization to XtrpCBl c strand DNA) was 6-8% of the total RNA synthe- K -[R -RO] [0-RO] [2] sized. The repressor fraction used has no activity without trypto- [RO] phan and does not affect either phage transcription or i strand where R = total repressor, 0 = total operator, [R - RO] = trp transcription. Longer preincubations of repressor with DNA free repressor, [O - RO I = free operator, and [RO] = re- (370) at the lowest repressor and DNA concentrations do not pressor-operator ] enhance repression, indicating that repressor-operator association complex. Equation [2 can be expressed as reaches equilibrium within 2 min at 00. K = 1 X [ 0]- (1 -X) [RI [3] where [01 = the operator concentration that saturates any given fraction X of repressor [X = (RO/R)]. For example, assumed to indicate the percent of total operators bound to when the operator concentration gives one-half saturation of repressor molecules. The total repressor concentration is deter- R, i.e., when RO = 1/2 R and X = '/2, equation [3] becomes mined from the plateau values in experiments in which the operator concentration is increased until the concentration of K = [01/2 - 1/2[R] [4] repressed operators reaches a plateau.