Cyclic AMP as a modulator of polarity in polycistronic transcriptional units.

Abstract

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...

Signal integration in the galactose network of Escherichia coli

“…GalE and GalT are also involved in internal inducer synthesis, when glucose is limited and extracellular galactose is not available (Death and Ferenci, 1994). Previous results indicate that transcription from the P1 galE promoter results in equimolar expression of the Gal enzymes, serving the catabolic requirements, while transcription from the P2galE promoter results in dis-coordinated expression (more expression of the promoter-proximal cistrons than the promoter-distal ones), which suites the biosynthetic requirements (Ullmann et al, 1979;Adhya, 2003;Semsey et al, 2006). Transcription of both P1galE and P2galE can be regulated by GalR and cAMP-CRP, while GalS affects only P1 galE activity.…”

Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon

“…When cAMP levels are reduced by mutations (cya − ) or catabolite repression (glucose as carbon source), the levels of GalK and GalT decline (the GalK level, in particular), but the GalE level remains high, resulting in an approximately fourfold increase in the GalE-to-GalK ratio. We note, however, that there are minor disagreements as to how cAMP levels or carbon source affect galE expression (Ullmann et al 1979;Joseph et al 1981;Adhya 1987). The discoordinate expression is more severe in the absence of a functional cAMP-CRP complex and can be suppressed in glucose-grown cells or in cya − strains by the addition of cAMP to the growth medium.…”

“…Moreover, work on the galactose operon has revealed that the cAMP-CRP complex, directly or indirectly, influences synthesis of the products of this operon differentially (Ullmann et al 1979;Joseph et al 1981;Adhya 1987). The E. coli galETKM operon is unusual in two respects.…”

Aspects of the Regulation of Adenylate Cyclase Synthesis in Escherichia coli K12

“…In Escherichia cofi, cAMP is also involved as a modulator of catabolite repression: low cAMP levels are correlated with a severe reduction in the synthesis of enzymes involved in carbon catabolism. In addition, the CAMP-CAP complex is involved in transcription termination of several polycistronic operons (Ullmann et al, 1979;Guidi-Rontani et al, 1984). However, whereas the molecular mode of action of the CAMP-CAP complex seems relatively well understood (De Crombrugghe et a f .…”