Proceedings of the National Academy of Sciences Vol. 68, No. 1, pp. 215-218, January 1971 An Adenosine 3':5'-Cyclic Monophosphate-Binding Protein That Acts on the Transcription Process L. ERON*, R. ARDITTI*, G. ZUBAYt, S. CONNAWAY*, AND J. R. BECKWITH* * Department of Bacteriology and Immunology, Harvard Medical School, Boston, Massachusetts 02115; and t Department of Biology, Columbia University, New York, N.Y. Communicated by Boris Magasanik, November 13, 1970 ABSTRACT An assay system for the in vitro transcrip- on the origin of the phages as predicted by the Campbell tion of the lac operon is described. A protein factor (CAP) model. This information indicates that there are no bacterial and cyclic AMP, which are essential for lac expression in vivo, also stimulate lac transcription in vitro. genes in common between the two phages other than the lac genes. However, since the X and q80 chromosomes do recom- Until recently, the study of the expression and regulation of bine with each other (7, 10, 11), there is almost certainly some bacterial genes has depended on physiological and genetic genetic homology between the two phage chromosomes. To studies. In the last few years, the detection and purification of test the effect of this homology on RNA-DNA hybridization regulatory proteins has opened up the possibility for studying we have transcribed 080plac and Xplac DNAs with RNA regulatory effects in vitro [e.g., lac (1), X (2), T4 (3), T7 (4)]. polymerase and hybridized the resulting RNA to the separate In the case of bacteriophage genomes, studies on in vitro strands of XDNA and 08ODNA, respectively. The results transcription have concentrated on regions of the phage ge- show that there is only very little homology between the RNA nomes (e.g., early and late), for the most part, rather than spe- made from one phage DNA and the DNA of the other (Table cific genes. In bacterial systems, however, one must be able to 1). This homology never amounts to more than 1% of the detect the RNA transcription of a relatively very limited total counts incorporated into the trichloroacetic acid number of genes, usually those comprising an operon. This (TCA)-precipitable material. We expect, therefore, that means that ideally for in vitro studies, an assay system for essentially the only RNA made from, for instance, 080plac mRNA must be developed that can pick up specifically the which will hybridize to Xplac is lac RNA itself. mRNA species from a single operon. Here, we describe such a For most of our experiments, we have used 080plac DNA system for the lac operon. as a template for transcription, and the separated strands of Among many transducing phages isolated that carry the Xplac for hybridization. In vivo the lac genes (16), and pre- lac operon, two have been described which have no other genes sumably all other genes, are transcribed into a mRNA comple- in common than the lac genes. These two phages, Xplac 5 and mentary to only one of the two DNA strands. We have shown 480plac 1, have been used, in fact, to isolate pure lac DNA (5). Thus, an assay system for specifically detecting lac mRNA Aplac, lac exists, either through the use of the pure lac DNA or through A ____ J y z b2 attA int N immA R use of the two phage DNA molecules, one as a template for Aplac, RNA transcription and the other for RNA-DNA hybridiza- Aplac, I_ tion. In this paper we describe the use of the hybridization proce- dure for studying in vitro transcription of the lac operon. These 080plac, studies have allowed us to show that a protein factor (CAP) and cyclic AMP (cAMP), which are essential for lac expression A ---- J att80 a i N imm80 R in vivo, act on the transcription process. The study of regula- 80pl zay tory effects in vitro, specifically effects of lac repressor and iac ,80plac1,I promoter mutations, suggest that additional components FIG. 1. Direction of transcription of genes on lac transduc- may be necessary for accurate initiation of lac transcription. ing phages. The arrows indicate direction of transcription and The results also indicate some of the problems in dealing with are placed closest to that strand from which the RNA is trans- in vitro transcription of a DNA preparation that contains cribed. In vitro, the b2 region of X is transcribed from both strands genes other than the genes of interest. (6). A brief description of the origins of the two phages is pre- sented elsewhere (5). The 480pilc behaves like an int- phage, METHODS AND RESULTS suggesting deletion of some genes to the left of N. Although in The probable structures of Xplac 5 and 080plac 1 genomes are the 80plac all y-distal bacterial genes were removed in the con- shown in Fig. 1. These structures are based on genetic data and struction of the phage, there are probably still bacterial genes adjacent to the i gene. We do not know how much of the b2 Abbreviation: CAP, a protein factor required for the synthesis region is intact in Xplac5. For maps of X describing the markers, of fl-galactosidase in vitro whose activity depends on cyclic AMP see refs. 7-9. For convenience, the 4)80 marker notation is identical (cAMP). with that of X. The iinm8O notation refers to o80 immunity and No reprints of this paper will be available in the USA. imm X to X immunity region. 215 Downloaded by guest on September 30, 2021 216 Biochemistry: Eron et al. Proc. Nat. Acad. Sci. USA that with q80plac, it is the 080plaCH strand and with Xplac, The failure to observe high levels of correct transcription of the XplaCL strand that is complementary to lac mRNA made the lac operon was not unexpected. Evidence from genetic and in vivo (5). One of the criteria, then, for determining whether physiological studies have indicated that for correct tran- the lac genes are being transcribed accurately in vitro is scription of the lac operon, in addition to RNA polymerase, whether the RNA made from 080plac is complementary to cAMP and a protein factor may be necessary (18-23). We the XplaCL strand only; i.e., asymmetric transcription. The have described the partial purification of a protein factor data in line 2 of Table 2 show that transcription of lac genes in (CAP) (22) which is necessary for the synthesis of fl-galactosi- 480plac DNA occurs from both strands, predominantly from dase in a crude in vitro system (15). The activity of this pro- the incorrect strand! This result is not unpredicted if one tein depends on the presence of cAMP. Emmer et al. (23) have considers the structure of the phage genome as outlined in described a similar protein. Our protein preparation binds Fig. 1. Transcription from the 480 "N" gene should be ini- cAMP with a constant of approximately 3 X 105 M. tiated in this system (17) and proceed onto the incorrect When CAP and cyclic AMP are included in the transcription strand of the lac region. [We show elsewhere that an RNA reaction mixture, up to a 6-fold stimulation of transcription termination factor, p, which eliminates transcription of the from the correct strand of the lac region is observed (Table 2, region to the left of N (17), does the same to the incorrect lines 2-5). At the concentrations used, the reaction is still transcription of the lac genes seen here (15).] linear with CAP concentration. At the same time as tran- scription from the correct strand of lac is stimulated, tran- scription from the opposite strand is depressed. This depres- TABLE 1. Complementarity of RNA transcribed from one lac sion may be due to competition between RNA polymerase transducing phuge DNA uith the heterologous wild molecules from type DNA transcribing opposite strands. phase The expected stimulation of lac transcription seen with and CAP is not due to a Cpm hybridized to cyclic-AMP generalized stimulation of transcription (total counts incorporated are the same) nor Template XL XH 48OL 480H probably to stimulation of transcription of parts of the 480 of the strand. The 080plac 147 (0.4) 165 (0.4) 10,000 (25) 17,400 (43) portions 480plaCH ratio of counts tran- Xplac ... ... 200 (0.5) 305 (0.7) scribed from 080plac which hybridized to 480L and 480H strands was identical whether or not CAP was present (Table The figures in parentheses are the % of total input counts 3). (40,000 cpm). The RNA polymerase used in these experiments includes Phage lysates were prepared as follows: 106-107 phage particles the a protein, which affects the template specificity of the were plated onto TYE plates (12) with M top agar (12) (diluted enzyme (3). The stimulation of lac transcription seen with 20% with distilled water) containing 2 X 108 bacteria of a lac CAP and cyclic AMP is completely dependent on the presence deletion strain (M182). After overnight incubation at 370C, of a factor in the transcription reaction mixture (Table 2, 1-2 ml of broth was added to each plate and left at room tem- lines 1 and 5). perature for an additional hour. The surface of the plates were The specificity of the effect of CAP and cyclic AMP on lac then scraped and the mixture of phage, bacteria, and agar was transcription suggests that transcription is proceeding in vitro treated with chloroform and centrifuged twice at 6000 rpm for 15 min. The as it does in vivo. A further criterion to establish this is to supernatant containing the phage was concentrated demonstrate in a Spinco ultracentrifuge at 22,000 rpm for 2.5 hr.