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Transcriptional Units Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3194-3197, July 1979 Biochemistry Cyclic AMP as a modulator of polarity in polycistronic transcriptional units (positive regulation/rho factor/lactose and galactose operons/catabolite repression) AGNES ULLMANNt, EVELYNE JOSEPHt, AND ANTOINE DANCHINt tUnite de Biochimie Cellulaire, Institut Pasteur, 75724 Paris Cedex 15, France; and tInstitut de Biologie Physico-Chimique, 75005 Paris, France Communicated by Frangois Jacob, April 16, 1979 ABSTRACT The degree of natural polarity in the lactose scribed by Watekam et al. (7, 8) in sonicated bacterial extracts. and galactose operons of Escherichia coli is affected by aden- One unit is the amount of enzyme that converts 1 nmol of osine 3',5'-cyclic monophosphate (cAMP). This effect, mediated substrate per min at 280C (except for UDPGal epimerase, for by the cAMP receptor protein, is exerted at sites distinct from the promoter. Experiments performed with a mutant bearing which the assay temperature was 220C). a thermosensitive rho factor activity indicate that cAMP relieves Reagents and Enzymes. They were obtained from the fol- polarity by interfering with transcription termination. Con- lowing companies: trimethoprim from Calbiochem; all radio- flicting results in the literature concerning the role of cAMP active products from Amersham; isopropyl-f3-D-thiogalactoside receptor protein and cAMP in galactose operon expression can (IPTG), D-fucose, cAMP, UDPglucose dehydrogenase, and all be reconciled by the finding that cAMP stimulates the expres- substrates from Sigma; and all other chemicals from Merck. sion of operator distal genes without significantly affecting the proximal genes. Therefore, it appears necessary to reevaluate the classification o(the galactose operon as exhibiting cAMP- RESULTS mediated catabolite repression at the level of transcription Natural Polarity in Lactose Operon. Nishi and Zabin have initiation. shown (9) that under normal growth conditions, f3-galactosidase (the first enzyme of the lac operon) is produced in excess with In prokaryotes, protein synthesis involves translation initiation respect to thiogalactoside transacetylase (the last enzyme of the at the beginning of each cistron of a polycistronic mRNA. A operon). In addition, they found that this natural polarity is salient feature of prokaryotic gene expression is the phenom- temperature dependent: the ratio of 13-galactosidase to tran- enon of polarity-i.e., the reduced expression of promoter distal sacetylase increases at higher temperatures. In a recent paper genes with respect to the proximal genes. It is generally believed (10) we showed that polarity is markedly enhanced when the that polarity is the result of premature termination of tran- cells are grown in the presence of trimethoprim, an inhibitor scription. One protein at least, the rho factor (1), is known to of the one-carbon pool metabolism (11). act in transcription termination, but its action is probably only The temperature dependence of the polarity brought about one aspect of the whole process (2). In this paper, we present by trimethoprim is even more pronounced than is the natural evidence suggesting that cyclic AMP (cAMP) and its receptor polarity. As can be seen in Fig. 1, natural polarity is significantly protein (CAP) act as antipolar effectors in the lactose and ga- increased at 40'C with respect to 300C, and this effect is further lactose operons. enhanced by trimethoprim. On the other hand, at 30'C, par- allel with the decrease in natural polarity, no effect of tri- MATERIALS AND METHODS methoprim can be detected. Furthermore, cAMP added to the Strains and Growth Conditions. The Escherichia coli K-12 growth medium completely abolishes polarity at all tempera- strains used throughout this work were CA8306 (cyaA); tures. To avoid interference with possible variations in the level CA8307 (crp) (generously given to us by Jon Beckwith); of intracellular cAMP, we performed the experiment shown CA8306 Lac+, a spontaneous high-level Lac+ pseudorevertant in Fig. 1 with a Lac+ pseudorevertant [presumed to carry a of CA8306 (mutant yield 10-10); CA8306 L8UV5, constructed mutation in the lac promoter (12)] of a strain carrying a deletion by transducing strain CA8306 to Lac+ with a P1 phage lysate in the adenylate cyclase gene (cyaA); however, similar results grown on L8UV5; CA8306 crp*, a Strs derivative (constructed were obtained with several cya + or cya strains (data not by Jacques Daniel) of strain CA8404 (cyaArp4) (obtained from shown). Because the overall expression of the lac operon is Jon Beckwith)-all derived from wild-type strain 3000. PP7811 strongly stimulated by cAMP (see Table 1), it could be argued and PP7812 isogenic F- argH his strains, except for the rho ts15 that the antipolar effect of cAMP might be somehow related allele (3), were constructed for this work. Strains were grown to the stimulation of transcription initiation. Therefore, the at 37°C in 63 minimal medium (4) supplemented with the re- same type of experiment was performed with the same cyaA quired amino acids, thiamine, and glucose as a carbon strain harboring, in addition, the cAMP-independent L8UV5 source. lac promoter. As shown in Fig. 2, the results are practically Enzymatic Assays. ,B-Galactosidase (EC 3.2.1.23) was identical to those reported above. Therefore, it can be con- assayed according to Pardee et al. (5) and thiogalactoside cluded that cAMP, besides its stimulatory effect at the level of transacetylase (EC 2.3.1.18) as described by Leive and Kollin transcription initiation, acts by relieving polarity in the lactose (6), both in toluenized bacterial suspensions. UDPgalactose operon. (UDPGal) epimerase (EC 5.1.3.2), uridyl transferase (EC Involvement of CAP in Antipolar Effect of cAMP. The 2.7.7.12), and galactokinase (EC 2.7.1.6) were assayed as de- well-known effect of cAMP as an antagonist of catabolite re- pression is mediated by its receptor protein, CAP (13), encoded The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: cAMP, adenosine 3',5'-cyclic monophosphate; CAP, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate cAMP receptor protein; UDPGal, uridine diphosphogalactose; IPTG, this fact. isopropyl-fl-D-thiogalactoside. 3194 Downloaded by guest on October 2, 2021 Biochemistry: Ullmann et al. Proc. Natl. Acad. Sci. USA 76 (1979) 3195 400C 400C -cAMP +cAMP -cAMP +cAMP Vta a, m Cu +W 100 W 100 Cu cCA 3700 c T0) -0 v7 Cu 300C 370C 3 c0 370C 400C 3O00 370C 4000 °0 50 3000C 50IF" IO FIG. 1. Effect of cAMP on polarity in the lactose operon: strain FIG. 2. Effect of cAMP on polarity in the lactose operon: strain CA8306 Lac+. To exponential cultures grown at 300C,370C, or 400C, CA8306 L8UV5. Experimental conditions were the same as described inducer (1 mM IPTG) alone (l) or inducer plus trimethoprim (0.5 in the legend of Fig. 1. ,ug/ml) (0) were added. After 1.5 generations ofgrowth [representing a mass increase of about 150 gg (dry weight) of bacteria per ml] tation that trimethoprim-induced polarity in the lactose operon 3-galactosidase and thiogalactoside transacetylase activities were is, at least partially, mediated by the rho protein. determined. Shown are rates of the ratios of differential synthesis of Polarity in Galactose Operon. The galactose operon of E. the enzymes. In a parallel experiment (Right), 5 mM cAMP was added together with the inducer. coli consists of three structural genes, galE, galT, and galK in this order, coding for a UDPGal epimerase, a uridyl transferase, by the crp gene. In order to know whether CAP was required and a galactokinase, respectively. The controlling sites are lo- for the antipolar effect of cAMP we made use of a specific crp cated at the galE end (16). Conflicting data exist concerning mutant (crp*) isolated as a pleiotropic carbohydrate-positive the control of this operon. Although gal operon expression is revertant in a cyaA background (14) and able to express cata- virtually the same in crp and cya mutants as in the wild-type bolite-sensitive operons in the absence of cAMP. Table 1 shows strain (17), galactokinase synthesis is stimulated by cAMP (18). that in this mutant natural and trimethoprim-induced polarity In addition, efficient in vitro expression of the operon requires are reduced or abolished whether in the absence or presence cAMP and CAP (19). These findings led Musso et al. (20) to of cAMP. This result suggests that the antipolar effect of cAMP suggest the existence of a dual control for transcription of the in the lac operon is mediated by CAP. galactose operon by cAMP and its receptor protein at two in- Involvement of Rho Factor in Lactose Operon Polarity. terspersed promoters. It is difficult to decide from in vivo experiments whether po- Most of the conclusions summarized above concerning the larity results from events occurring at the translation or the control of gal operon expression are based either on in vitro transcription level. Translation might certainly be involved, but studies or on in vivo measurements of the activity of a single one would not expect a gradient from the promoter proximal enzyme, galactokinase. Because galactokinase is encoded by cistron toward the most distal one because it would require a the operator distal gene, variations in its rate of synthesis might parallel gradient in the efficiency of translation initiation. Po- not necessarily be related to transcription initiation control, but larity is usually considered to be due to premature termination could just as well reflect a polar effect. Therefore, we measured of transcription (1) or endonucleolytic attack of the mRNA (15) the rates of synthesis of all three enzymes of the gal operon in or both. Premature termination of transcription might be wild-type strains and in cya, crp, or rho thermosensitive mu- caused by a lack of affinity of RNA polymerase for specific tants in the absence or in the presence of cAMP.
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