We have studied the correlation between the activities of adenylate cyclase (ATP pyrophosphatelyase-(cyclizing); EC 4.6.1.1) and in vivo rates of synthesis and intracellular concentrations of adenosine 3',5' cyclic monophosphate (cAMP) under various growth conditions in wild-type Escherichia coli and in mutants lacking or overproducing the cAMP receptor protein (CAP). We showed that when wild-type bacteria are grown in the presence of a variety of carbon sources the intracellular concentrations of cAMP are inversely related to the adenylate cyclase activities determined in permeabilized cells, suggesting that the carbon source-dependent modulation of cAMP levels is not directly related to the regulation of adenylate cyclase activity. In mutants lacking functional CAP (crp) the in vivo rates of cAMP synthesis are several hundred-fold higher than in the wild-type parent without a parallel increase of adenylate cyclase activities. In a strain carrying multiple copies of the crp gene and overproducing CAP the activity of adenylate cyclase is severely inhibited, although the in vivo rate of cAMP synthesis is similar to the parental strain. We interpret these results as indicating that CAP controls mainly the activity rather than the synthesis of adenylate cyclase.
The degree of natural polarity in the lactose and galactose operons of Escherichia coli is affected by adenosine 3',5'-cyclic monophosphate (cAMP). This effect, mediated by the cAMP receptor protein, is exerted at sites distinct from the promoter. Experiments performed with a mutant bearing a thermosensitive rho factor activity indicate that cAMP relieves polarity by interfering with transcription termination. Conflicting results in the literature concerning the role of cAMP receptor protein and cAMP in galactose operon expression can be reconciled by the finding that cAMP stimulates the expression of operator distal genes without significantly affecting the proximal genes. Therefore, it appears necessary to reevaluate the classification o(the galactose operon as exhibiting cAMPmediated catabolite repression at the level of transcription initiation.In prokaryotes, protein synthesis involves translation initiation at the beginning of each cistron of a polycistronic mRNA. A salient feature of prokaryotic gene expression is the phenomenon of polarity-i.e., the reduced expression of promoter distal genes with respect to the proximal genes. It is generally believed that polarity is the result of premature termination of transcription. One protein at least, the rho factor (1), is known to act in transcription termination, but its action is probably only one aspect of the whole process (2). In this paper, we present evidence suggesting that cyclic AMP (cAMP) and its receptor protein (CAP) act as antipolar effectors in the lactose and galactose operons. One unit is the amount of enzyme that converts 1 nmol of substrate per min at 280C (except for UDPGal epimerase, for which the assay temperature was 220C). MATERIALS AND METHODSReagents and Enzymes. They were obtained from the following companies: trimethoprim from Calbiochem; all radioactive products from Amersham; isopropyl-f3-D-thiogalactoside (IPTG), D-fucose, cAMP, UDPglucose dehydrogenase, and all substrates from Sigma; and all other chemicals from Merck. RESULTS Natural Polarity in Lactose Operon. Nishi and Zabin haveshown (9) that under normal growth conditions, f3-galactosidase (the first enzyme of the lac operon) is produced in excess with respect to thiogalactoside transacetylase (the last enzyme of the operon). In addition, they found that this natural polarity is temperature dependent: the ratio of 13-galactosidase to transacetylase increases at higher temperatures. In a recent paper (10) we showed that polarity is markedly enhanced when the cells are grown in the presence of trimethoprim, an inhibitor of the one-carbon pool metabolism (11).The temperature dependence of the polarity brought about by trimethoprim is even more pronounced than is the natural polarity. As can be seen in Fig. 1, natural polarity is significantly increased at 40'C with respect to 300C, and this effect is further enhanced by trimethoprim. On the other hand, at 30'C, parallel with the decrease in natural polarity, no effect of trimethoprim can be detected. Furthermore, cAMP...
2-ketobutyrate and its analogues were found to inhibit strongly and transiently the rate of beta-galactosidase synthesis in Escherichia coli K12. This effect was ascribed to a strong and transient inhibition of the adenylate cyclase activity. By using pts mutants, we showed, in agreement with our previous results (Daniel et al. 1983), that the likely target of 2-ketobutyrate and its analogues is the phosphoenolpyruvate: glycose phosphotransferase transport system (PTS). Furthermore, evidence for such a cascade effect caused by 2-ketobutyrate and its analogues allowed us to corroborate our previous proposal (Daniel et al. 1983) that 2-ketobutyrate, a precursor of isoleucine, acts as an E. coli alarmone monitoring the passage from anaerobic to aerobic growth conditions.
Eucaryotic and procaryotic organisms differ in two aspects of their translation machinery: polycistronic messengers are expressed as a sequence of individual proteins only in procaryotes, and the initiation of protein synthesis proceeds with an initiator tRNA which is found to be modified (formylated) in procaryotes and not in eucaryotes. In the present study, we show that formylation is required in vivo for the coordinate expression of the Escherichia coli lactose operon. Our experiments are consistent with a translation mechanism using dissociated ribosomes at the 5' end of the mRNA in a reaction that is only weakly dependent on enzyme of the lactose operon of Escherichia 453 on July 8, 2020 by guest
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