we extracted DNA from three types of 080 phages transducing

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 68, No. 8, pp , August 1971 Mechanism of Initiation and Repression of In Vitro Transcription of the Lac Operon of Escherichia coli (cyclic AMP/RNA polymerase/sigma factor/rho factor/camp-binding protein) LARRY ERON* AND RICARDO BLOCK * Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115; and The Biological Laboratories, Harvard University, Cambridge, Mass Communicated by Boris Magasanik, May 21, 1971 ABSTRACT A cyclic AMP-binding protein (CAP protein), cyclic AMP, and RNA polymerase holoenzyme are shown to initiate lac transcription at the lac promoter. Lac repressor appears to control transcription by preventing RNA polymerase and/or CAP protein from binding to the lac promoter. Results support the idea that the lac promoter is composed of two sites that interact with CAP protein and RNA polymerase holoenzyme. The promoter can be altered by mutation so that holoenzyme alone can initiate lac transcription correctly. The expression of the lactose (lac) and other operons of Escherichia coli subject to catabolite repression requires adenosine 3':5'-cyclic monophosphate (cyclic AMP) (1, 2) and a cyclic AMP-binding protein (CAP protein) (3, 4). In a purified in vitro transcription system (5, 6), cyclic AMP and CAP protein have been shown to act at the level of transcription, and the effect is dependent on the presence of a, a subunit of RNA polymerase necessary for initiation of transcription at phage promoters (7); this result indicates that CAP protein acts in a manner different from a. Surprisingly, however, in this system, which employs as template DNA extracted from a transducing phage (480plac) carrying lac in place of the early genes of the phage in the orientation shown in Fig. 1, effects of lac repressor and lac promoter mutations on lac transcription could not be demonstrated (6). However, control of lac transcription by lac repressor has been obtained (8, 27) by use of a two-step hybridization assay of RNA synthesized from a phage template, similar to 480dlac in Fig. 1, carrying lac in place of the late genes of the phage in an orientation opposite to that in 080 plac. Therefore, we decided to reinvestigate the transcription of lac from the 4)80dtac template by a simple one-step hybridization assay (5, 6). Using a simple one-step hybridization procedure (5, 6), we confirm that in a purified in vitro transcription system, lac operon transcription is stimulated asymmetrically from the correct DNA strand by cyclic AMP and a cyclic AMPbinding protein (CAP protein), and is controlled by repressor (8, 27). This effect is dependent on the presence of a factor, indicating that CAP protein acts in a manner different from a. In addition, we show that the transcription initiates at the lac promoter, since it is affected by lac promoter mutants on the transcription template. One of these mutants, pr uv-5, allows initiation of lac transcription in the absence of CAP Abbreviation: CAP protein, a cyclic AMP-binding protein, referred to elsewhere as CRP (8, 27).; lac, the lactose operon of E. coli protein, although it retains its requirement for a. Apparently, the lac promoter has been altered so that it can initiate in the absence of transcription factors other than a. Our results suggest that the lac repressor acts by preventing RNA polymerase and/or CAP protein from binding to the lac promoter. For template in the purified in vitro transcription system, we extracted DNA from three types of 080 phages transducing lac: )80dlaci, )80dlac,11, and 480plac (see Fig. 1). The first two carry lac in an orientation opposite to that of 4)80plac, transcribed in vivo from the L-strand, while on 480- plac, lac is transcribed from the H-strand (9). To assay lac transcription, RNA synthesized from these templates is directly hybridized to separated strands of Xplac DNA. Only lac RNA should anneal to Xplac DNA, since there is less than 0.5% homology between )80 RNA and XDNA (5, 6). Although the three phages used as template DNA carry bacterial genes adjacent to lac (5), this should not interfere with the assay of lac sequences, because all non-lac bacterial genes have been deleted in the pxlac phage phage (9). Furthermore, correctly initiated lac RNA should anneal to the XplacL las.~4-0s~iik A--- y z i att8 uint N imm80 R 0.0dbc,,, A_ ; eother bacterial gems A---J aft80i Z y V m8 R I $080PWI_ FIG. 1. Direction of transcription of genes on lac transducing phages. The arrows indicate the direction of transcription and are placed closest to that strand from which the RNA is transcribed (11, 12). The subscripts H and L refer to the heavy and light DNA strands after CsCl equilibrium density gradient ultracentrifugation after annealing by poly(ru, G) (13). The origin and marker notation of the phages are described elsewhere (6, 9, 10). 080dlaciii was derived from the X-+080 hybrid phage (20), Xh8Odlac (5), by recombination with 480. Lysogens of X h80dlac were plated overnight at 30'C with 10' 480/plate. The lysates were harvested and transduced into M182, alac deletion strain, on lactose minimal medium at 42'C. 50% of the lac+ transductants were lysogens of 080/080dlaciii. RNA synthesized in vitro from 4)80dlacixi has less than 0.5% homology with separated strands of XDNA (L.E., unpublished results). 480dlaci is an independently isolated defective transducing phage carrying lac in the same location and orientation as 080dlacil (21). 480plac is an infectious transducing phage carrying lac in a different location and orientation (see text and ref. 6).

2 Proc. PcRegulation Nat. Acad. Sci. USA 68 (1971) of Lac Transcription 1829 TABLE 1. #-galactosidase synthesis in vitro directed by phage templates carrying lac A420 with CAP A420 with CAP protein + S-30 protein- S-30 - cyclic +cyclic CAP CAP Template AMP AMP protein protein 080dlacilx dlaciii p dlacjIipruv_ dlacx q0801acil plac The experimental procedure, 8-30 preparation, reaction mixture, and fl-galactosidase assay are described in detail elsewhere (18). Reaction mixtures (75 sl) contained, where indicated, cyclic AMP (0..5 mm) and CAP protein (25,l), purified 200-fold, the gift of G. Zubay (3). CAP protein (2 Ag/ml), purified to homogeneity (gift of W. Anderson, R. Perlman, and I. Pastan), behaved identically with the partially purified CAP protein. Although the pure CAP protein is referred to elsewhere as CRP (4, 8), we have adopted a unified nomenclature for clarity. Except where indicated in Fig. 2 and Tables 3 and 4, we have used partially purified CAP protein. fl-galactosidase synthesis is normalized to absorbance units at 420 nm of product formed per 200-,l reaction mixture incubated for 20 hr. Strains used to prepare S-30 fractions are RV, a lac deletion (X74) strain that is CAP protein +, and X7901, a Re-pro A-pro B deletion strain that is CAP protein-. The p' (the gift of M. Gottesman), pruv-5, and Li mutations are described in the text. strand only, since this is the strand transcribed in vivo (9). Using this assay system, we have examined the fidelity of lac transcription in vitro according to four criteria: (a) Lac RNA synthesis should be dependent on the presence of cyclic AMP and CAP protein (1-4), (b) should occur asymmetrically, that is, anneal to XplaCL strand only (9, 14), (c) should initiate at the la promoter (15, 16, 23), and (d) should be controlled by lac repressor (17). TABLE ~1 - "dump" Eproteiasa l RAPlmrae(/l protein (pg/mi) RNA Polymnerase (jig/mi) FIG. 2. Effect of CAP protein and RNA polymerase concentration on lac transcription. (a) Conditions were as in Table 2, except that pure CAP protein was used at the concentrations indicated with cyclic AMP (1 mm) and the indicated templates. ['HIRNA (45,000 cpm/tube) was annealed to XAPlaCL DNA. (b) Conditions were as in Table 2 except that RNA polymerase concentration was varied with the indicated templates. Pure CAP protein (7 pg/ml) and cyclic AMP (1 mm) were present. Total RNA synthesis was linear with RNA polymerase concentration, and the input of [3H]RNA was normalized to 45,000 cpm/tube and annealed to XplaCL DNA. Asymmetric stimulation of 1ac transcription by CAP protein and cyclic AMP The first two transducing phages, 080dlac1 and 080dlaci1, are efficient DNA templates for 0-galactosidase synthesis in a crude cell-free system (Table 1). In the purified transcription system, they produce essentially no lac RNA in thte absence of CAP protein and cyclic AMP (Table 2; Fig. 2) even in the absence of transcription termination factor p (11), indicating the absence of read-through in our system. When Effect of CAP protein, cyclic AMP, and a factor on lac transcription cpm Hybridized to Template CAP protein cyclic AMP XplaCL-XL XPlaCH-XH % lao RNA j,80dlaciii <0. 1 o80dlacili o80dlaciii , *a 080dlacirn (2)* 080dacilips , dlociiip" (4)* 4,80dacIiips (4)* 080dlacIijpruv , q080dlacjiipruv , dlaciiipruv_ (3)* [3HJ RNA is synthesized in a reaction mixture described in detail elsewhere (5, 6) with the following modifications: DNA (50 jg/ml), KC1 (120 mm), IPTG (1 mm), [3H]ATP (0.1 mm), and RNA polymerase (20 /Ag/ml), purified to homogeneity-either as core or holoenzyme from a rifampicin-resistant strain (gift of K. Weber). CAP protein (25 pl) and cyclic AMP (1 mm) were incubated for 1 min at 370C with the reaction mixture before the addition of RNA polymerase. The reaction was incubated 10 min at 370C, terminated, extracted, and hybridized (5, 6, 22). [3H]RNA (45,000 cpm/tube with holoenzyme polymerase; 3,500 cpm/tube with core polymerase) was annealed to separated strands of Xplac and xdna; the difference is expressed above; cpm annealing to X separated strands was always less than 0.5% of the [3HJRNA input. * % bac RNA is enclosed in brackets for experiments with core polymerase because it does not reflect correct initiation (see text).

3 1830 Microbiology: Eron and Blcck ai)._ I E~ C) P(pg/ml) FIG. 3. Effect of p on kac transcription from 080dlacill and 480plac templates. Conditions were as in Table 2, except that p (purified to homogeneity, free of RNAase III, the gift of N. Minkley) was added as indicated to reaction mixtures containing CAP protein (25 Ml) and cyclic AMP (1 mm). The addition of p produces a two- to three-fold depression of total RNA synthesis. [8H]RNA (in the absence of p, 60,000 cpm/tube for 080dlac,11 RNA and 50,000 cpm/tube for c80plac RNA; 25,000 and 20,000 cpm/tube, respectively, in the presence of excess p) was annealed to (-) XplaCL DNA and (0) XPlaCH DNA. CAP protein and cyclic AMP are added, lac transcription is stimulated in an asymmetric fashion from the correct DNA strand to maximal concentrations of 5% of the total RNA synthesized from the template. The above situation contrasts sharply with lac transcription from the 480plac template. As we have reported (5, 6), lac RNA synthesis from this template is stimulated asymmetric Hly by CAP protein and cyclic AMP, but is not subject to control by the lac promoter and repressor. The fact that transcription termination factor p decreases CAP protein-depandent lac transcription (see Fig. 3) suggests that lac transcription from this phage template is the result of transcription initiation in a nearby, similarly oriented gene, with subsequent read-through into the lac genes. In vivo studies in- TABLE 3. Repression of lac transcription Re- cpm Hybridized to % lac Template pressor IPTG XplacL-XL RNA 080dlacIix - + 3, dlaciii dkciii + + 2, ,80dlacilnpruv , dlacIIIpruv , dlaciiipruv , dlaciiip - + 3, dlaciiip' dlaciiipe + + 3,560 8 Conditions were as in Table 2, except that lac repressor (5 lug/ml, purified to homogeneity, a gift of T. Platt) was incubated 2 min at 37 C with thte template DNA in 50 mm KCl-10 mm MgC12 before addition of CAP protein. Pure CAP protein (7 /ug/ml) and cyclic AMP (1 mm) were incubated 1 min at 370C with the repressor-dna complex before RNA polymerase (20 lug/ml) was added. IPTG was added where indicated with CAP protein. [3H]RNA (45,000 cpm/tube) was annealed to XplaCL- DNA and XLDNA; the difference is expressed above. Proc. Nat. Acad. Sci. USA 68 (1971) CAP protein RNA Polymerase )( Holoenzyme Spl, o z y a LI FIG. 4. Model for the interaction of CAP protein and RNA polymerase holoenzyme with the lac promoter. Genes p and o refer to the lac promoter and operator regions. i codes for lac repressor, and z, y, and a for the lac enzymes. dicate that such read-through should not be subject to lac promoter control, and that repression might be less efficient (23). Furthermore, this phage is an extremely poor template for P-galactosidase synthesis in a crude, cell-free system (Table 1), although it produces normal levels of,b-galactosidase in lysogens in vivo (5). In addition, using DNA extracted from Xplac, a phage carrying lac in place of the phage late genes in an orientation opposite to that of 080plac, we could not demonstrate effects of cyclic AMP and CAP protein (6). These discrepancies may be due to the orientation and location of the lac genes with respect to other genes on the phage template. Lac transcription is initiated at the lac promoter Initiation of CAP protein- and cyclic AMP-dependent lac transcription occurs in vivo at the lac promoter (15, 16, 24). If the in vitro transcription system is behaving properly, lac transcription should initiate at the lac promoter also. To ascertain this, we have assayed lac transcription with various promoter mutations on the 480dlac template DNA. First, we have used L1, a small deletion of part of the promoter and the adjacent i gene that results in a 100-fold reduction in 3-galactosidase synthesis in vivo (24), and a 10- fold reduction in the crude, cell-free system (Table 1). In the purified transcription system, it produces from 3- to 10-fold less lac RNA, depending on the conditions (Fig. 2). Moreover, in vivo studies indicate that Li is insensitive to CAP protein and cyclic AMP (25). In the cell-free system, Li shows no dependence on cyclic AMP (Table 1). In the purified tratnscription system, only a slight stimulation of Li lac transcription occurs when pure CAP protein and cyclic AMP are added (Fig. 2). We have examined another category of lac promoter mutants termed "super-promoters" (8), because they produce 10- to 30-fold more p-galactosidase in the cell-free system (Table 1). One super-promoter, designated pruv-5, is a revertant of the lac promoter mutant L8, and is a second-site mutation within the lac promoter (26). While L8 produces 15-fold less,b-galactosidase in vivo (24), pruv-5 produces normal levels of 0-galactosidase. In addition, pruv-5 has lost its requirement for CAP protein and cyclic AMP both in vivo (26) and in the cell-free system (Zubay, unpublished results; Table 1). The other super-promoter, p', isolated as a revertant of a CAP protein- cell, retains a partial requirement for CAP protein and cyclic AMP (8). In the purified transcription system, lac RNA levels, synthesized from template DNA carrying super-promoter mutations, are elevated from 2- to 10-fold over wild-type depending on the conditions (Fig. 2). The striking feature about lac transcription from the 480- dkaciipruv-5 DNA template is that a substantial amount occurs in the absence of CAP protein (although CAP protein does stimulate it).

4 Proc. Nat. Acad. Sci. USA 68 (1971) Regulation of Lac Transcription 1831 TABLE 4. Effect of repressor on lac RNA synthesis by prebound RNA polymerase Preincubation mixture CAP Tube XTP-UTP protein + cyclic AMP Repressor IPTG cpm of cpm Hybridized to [3H]RNA/tube XplacL-XL % lac RNA , ,000 1, ,000 1, , , (before - 5, polymerase) The reaction mixture in tubes 1-3 was as in Table 2 with 480dtaciiips DNA (50.ug/ml) and rifampicin-sensitive RNA polymerase (20 /Ag/ml), except that UTP was omitted from the preincubation mixture. This mixture was incubated 10 min at 200C. Repressor (5 /g/ml), IPTG (1 mm), rifampicin (5 csg/ml), and finally UTP (0.15 mm) were then added and the reaction was further incubated 10 min at 370C. In tube 1, CAP protein and cyclic AMP were omitted from the preincubation mixture. The reaction mixture in tubes 4-6 was as above, except that all four nucleoside triphosphates were omitted from the preincubation, and were added after rifampicin. In tube 6, repressor was added before RNA polymerase. [3Hl RNA at the indicated input/tube was annealed to XPkaCL and XL DNA; the difference is expressed above. Since L1, pruv-5, and p8 control transcription in the purified system, we conclude that CAP protein and cyclic AMP stimulate lac transcription predominantly at the lac promoter with q80dlac DNA as template in the purified system. If this is true, then lac transcription from this template should not be depressed by the addition of transcription termination factor p, contrary to the case of the c80plac template (5), where lac transcription results from read-through as described in the previous section. Indeed, only a 10% decrease in lac transcription is observed in the presence of p with the 480dlac template, as compared to an 80% decrease with the 080plac template (Fig. 3). That p has little or no effect on lac transcription from the,80dlac template confirms that lac transcription from the 480dlac template initiates predominantly at the lac promoter. As we have shown previously (5, 6), a acts in a manner different from CAP protein. Core polymerase (without a) transcribes lac symmetrically and shows no requirement for cyclic AMP, indicating that it is not initiating correctly at the lac promoter (Table 2). Repression of lac transcription As others have shown (8, 27), when lac repressor is added to the transcription system, lac transcription is repressible from 50 to 95%, the mode being 75% (Table 3). This repression is completely reversible by the inducer, IPTG. Bacteriophage X repressor has been proposed to act by competing with RNA polymerase for binding to the X regulatory elements (12). Our results indicate that lac repressor acts in the same way (Table 4). RNA polymerase, CAP protein1, cyclic AMP, and lac p8 DNA are incubated in the absence of either one nucleoside triphosphate (to allow polymerase binding and initiation in tubes 2 and 3) or in the absence of all four nucleoside triphosphates (to allow polymerase binding, but not initiation in tubes 4 and 5). After this incubation, rifampicin is added to inhibit further initiation. Repressor is then added, either with or without IPTG. The results in Table 4 indicate that repressor cannot block lac transcription once CAP protein and RNA polymerase binding or initiation has occurred, while, if added before binding, it blocks the formation of an initiation complex. This suggests that repressor acts by preventing the binding of CAP protein and/or RNA polymerase to the lac promoter. DISCUSSION The lac promoter mutation Li supports the idea that the lac promoter is composed of at least two sites (27). Since strains with Li produce,-galactosidase at an equally low rate in the presence and absence of CAP protein, it appears that the site of the CAP protein effect has been deleted (Fig. 4). However, the remaining low level of lac expression, which cannot be due to read-through from another gene (23), indicates that the part of the promoter that is intact can still serve as a weak transcription initiation site. This part of the promoter may be the site where RNA polymerase holoenzyme (core plus a) normally binds (Fig. 4), and CAP protein would greatly enhance this binding by interacting with the adjacent region. On this model, super-promoter pruv-5 would alter the holoenzyme binding site so that the promoter can bind holoenzyme in the absence of CAP protein. We thank Dr. Jonathan Beckwith for his advice and helpful suggestions and Dr. Walter Gilbert for critical examination of this manuscript. We also thank Penelope Wood for her excellent technical assistance, G. Zubay and R. Perlman, W. Anderson, and I. Pastan for CAP protein, M. Gottesman for the p" mutant, N. Minkley for p, K. Weber for RNA polymerase, and T. Platt for repressor. This work was supported in part by grants from the National Science Foundation, The American Cancer Society, and the Jane Coffin Childs Memorial Fund for Medical Research to J. Beckwith, and in part from the NIH Grant GM09541 to J. D. Watson. R. B. received a scholarship from the Instituto Nacional de la Investigacion Cientifica, Mexico. 1. Perlman, R. L., and I. Pastan, J. Biol. Chem., 243, 5420 (1969). 2. Ullman, A., and J. Monod, FEBS Lett., 2, 57 (1968). 3. Zubay, G., D. Schwartz, and J. Beckwith, Proc. Nat. Acad. Sci. USA, 66, 104 (1970). 4. Emmer, M., B. decrombrugghe, I. Pastan, and R. Perlman, Proc. Nat. Acad. Sci. USA, 66, 480 (1970). 5. Arditti, R. R., L. Eron, G. Zubay, G. Tocchini-Valentini, S. Connaway, and J. R. Beckwith, Cold Spring Harbor Symp. Quant. Biol., 35, 437 (1970). 6. Eron, L., R. Arditti, G. Zubay, S. 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