Proc. Nat. Acad. Sci. USA Vol. 68, No. 8, pp. 1828-1832, 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. 02138 Communicated by Boris Magasanik, May 21, 1971 ABSTRACT A cyclic AMP-binding protein (CAP pro- protein, although it retains its requirement for a. Ap- tein), cyclic AMP, and RNA polymerase holoenzyme are shown to initiate lac transcription at the lac promoter. parently, the lac promoter has been altered so that it can Lac repressor appears to control transcription by prevent- initiate in the absence of transcription factors other than a. ing RNA polymerase and/or CAP protein from binding to Our results suggest that the lac repressor acts by preventing the lac promoter. Results support the idea that the lac RNA polymerase and/or CAP protein from binding to the promoter is composed of two sites that interact with CAP lac promoter. protein and RNA polymerase holoenzyme. The promoter can be altered by mutation so that holoenzyme alone can For template in the purified in vitro transcription system, initiate lac transcription correctly. we extracted DNA from three types of 080 phages trans- ducing lac: )80dlaci, )80dlac,11, and 480plac (see Fig. 1). The expression of the lactose (lac) and other operons of The first two carry lac in an orientation opposite to that of Escherichia coli subject to catabolite repression requires 4)80plac, transcribed in vivo from the L-strand, while on 480- adenosine 3':5'-cyclic monophosphate (cyclic AMP) (1, 2) plac, lac is transcribed from the H-strand (9). To assay and a cyclic AMP-binding protein (CAP protein) (3, 4). In lac transcription, RNA synthesized from these templates is a purified in vitro transcription system (5, 6), cyclic AMP directly hybridized to separated strands of Xplac DNA. Only and CAP protein have been shown to act at the level of lac RNA should anneal to Xplac DNA, since there is less than transcription, and the effect is dependent on the presence of 0.5% homology between )80 RNA and XDNA (5, 6). Al- a, a subunit of RNA polymerase necessary for initiation of though the three phages used as template DNA carry bacterial transcription at phage promoters (7); this result indicates genes adjacent to lac (5), this should not interfere with the that CAP protein acts in a manner different from a. assay of lac sequences, because all non-lac bacterial genes have Surprisingly, however, in this system, which employs as been deleted in the pXlac phage phage (9). Furthermore, template DNA extracted from a transducing phage (480plac) correctly initiated lac RNA should anneal to the XplacL carrying lac in place of the early genes of the phage in the las orientation shown in Fig. 1, effects of lac repressor and lac .~4 - promoter mutations on lac transcription could not be dem- 0S~iIKA--- y z i att8 uint N imm80 R onstrated (6). However, control of lac transcription by lac 0.0dbc,, , A_ ; repressor has been obtained (8, 27) by use of a two-step hy- eother bacterial gems bridization assay of RNA synthesized from a phage template, A---J aft80i Z y V m8 R similar to 480dlac in Fig. 1, carrying lac in place of the late $080PWI_ I genes of the phage in an orientation opposite to that in 080 plac. Therefore, we decided to reinvestigate the transcription FIG. 1. Direction of transcription of genes on lac transducing of lac from the 4)80dtac template by a simple one-step hy- phages. The arrows indicate the direction of transcription and bridization assay (5, 6). are placed closest to that strand from which the RNA is trans- Using a simple one-step hybridization procedure (5, 6), we cribed (11, 12). The subscripts H and L refer to the heavy and light DNA strands after CsCl equilibrium density gradient ultra- confirm that in a purified in vitro transcription system, lac centrifugation after annealing by poly(rU, G) (13). The origin operon transcription is stimulated asymmetrically from the and marker notation of the phages are described elsewhere (6, 9, correct DNA strand by cyclic AMP and a cyclic AMP- 10). 080dlaciii was derived from the X-+080 hybrid phage (20), binding protein (CAP protein), and is controlled by repressor Xh8Odlac (5), by recombination with 480. Lysogens of X h80dlac (8, 27). This effect is dependent on the presence of a factor, were plated overnight at 30'C with 10' 480/plate. The lysates indicating that CAP protein acts in a manner different from were harvested and transduced into M182, alac deletion strain, on a. In addition, we show that the transcription initiates at the lactose minimal medium at 42'C. 50% of the lac+ transductants lac promoter, since it is affected by lac promoter mutants on were lysogens of 080/080dlaciii. RNA synthesized in vitro from the transcription template. One of these mutants, pr uv-5, 4)80dlacixi has less than 0.5% homology with separated strands allows initiation of lac transcription in the absence of CAP of XDNA (L.E., unpublished results). 480dlaci is an independently isolated defective transducing phage carrying lac in the same lo- Abbreviation: CAP protein, a cyclic AMP-binding protein, re- cation and orientation as 080dlacil (21). 480plac is an infectious ferred to elsewhere as CRP (8, 27).; lac, the lactose operon of E. transducing phage carrying lac in a different location and orienta- coli. tion (see text and ref. 6). 1828 Downloaded by guest on September 27, 2021 Proc.PcRegulationNat. 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 4000 ~1- 080dlacilx 0.04 0.35 "dump" 480dlaciii p 0.06 10.00 0.44 7.15 480dlacjIipruv_5 18.10 34.60 20.20 30.90 080dlacx 0.07 0.73 q0801aciL1 0.07 0.06 - 2000- 080plac 0.01 0.06 - Eproteiasa l RAPlmrae(/l The experimental procedure, 8-30 preparation, reaction mix- ture, and fl-galactosidase assay are described in detail elsewhere (18). Reaction mixtures (75 sl) contained, where indicated, cyclic 1 5 10 15 10 20 40 AMP (0..5 mM) and CAP protein (25 ,l), purified 200-fold, the protein (pg/mi) RNA Polymnerase (jig/mi) gift of G. Zubay (3). CAP protein (2 Ag/ml), purified to ho- mogeneity (gift of W. Anderson, R. Perlman, and I. Pastan), FIG. 2. Effect of CAP protein and RNA polymerase con- behaved identically with the partially purified CAP protein. centration on lac transcription. (a) Conditions were as in Table Although the pure CAP protein is referred to elsewhere as CRP 2, except that pure CAP protein was used at the concentrations (4, 8), we have adopted a unified nomenclature for clarity. Except indicated with cyclic AMP (1 mM) and the indicated templates. where indicated in Fig. 2 and Tables 3 and 4, we have used ['HIRNA (45,000 cpm/tube) was annealed to XAPlaCL DNA. (b) partially purified CAP protein. fl-galactosidase synthesis is Conditions were as in Table 2 except that RNA polymerase con- normalized to absorbance units at 420 nm of product formed per centration was varied with the indicated templates. Pure CAP 200-,l reaction mixture incubated for 20 hr. Strains used to pre- protein (7 pg/ml) and cyclic AMP (1 mM) were present. Total pare S-30 fractions are RV, a lac deletion (X74) strain that is RNA synthesis was linear with RNA polymerase concentration, CAP protein +, and X7901, a Re-pro A-pro B deletion strain that and the input of [3H]RNA was normalized to 45,000 cpm/tube is CAP protein-. The p' (the gift of M. Gottesman), pruv-5, and and annealed to XplaCL DNA. Li mutations are described in the text. Asymmetric stimulation of 1ac transcription by CAP strand only, since this is the strand transcribed in vivo (9). protein and cyclic AMP Using this assay system, we have examined the fidelity of lac The first two transducing phages, 080dlac1 and 080dlaci1, transcription in vitro according to four criteria: (a) Lac RNA are efficient DNA templates for 0-galactosidase synthesis in synthesis should be dependent on the presence of cyclic AMP a crude cell-free system (Table 1). In the purified transcrip- and CAP protein (1-4), (b) should occur asymmetrically, that tion system, they produce essentially no lac RNA in thte ab- is, anneal to XplaCL strand only (9, 14), (c) should initiate at the sence of CAP protein and cyclic AMP (Table 2; Fig. 2) even la promoter (15, 16, 23), and (d) should be controlled by lac in the absence of transcription termination factor p (11), repressor (17). indicating the absence of read-through in our system. When TABLE 2. 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 + 1 22 <0.1 o80dlacili + - + 189 58 0.4 o80dlaciii + + + 2,289 124 *a 080dlacirn + + - 85 57 (2)* 080dacilips + + + 3,210 101 7 4080dlociiip" + + - 150 132 (4)* 4,80dacIiips + - - 147 125 (4)* 080dlacIijpruv-5 + - + 1,708 73 4 q080dlacjiipruv-5 + + + 3, 94.5 54 9 080dlaciiipruv_5 + + - 108 65 (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 holo- enzyme from a rifampicin-resistant strain (gift of K.