Action of CAP on the malT promoter in vitro

Abstract

DNase I footprinting experiments demonstrated that CAP, the cyclic AMP receptor protein of Escherichia coli, binds around position -70 at the promoter of malT, the positive regulator gene of the maltose regulon. The binding of CAP in the presence of cyclic AMP favored the subsequent specific binding of RNA polymerase. Initiation of malT transcription in vitro displayed an absolute requirement for CAP at all tested RNA polymerase concentrations. However this was not the case with a mutant promoter (malTpl), which leads to CAPindependent malT expression in vivo. In that case an effect of CAP was seen only at the lower concentrations of RNA polymerase. These results, which suggest that CAP stimulates malT expression by promoting the binding of polymerase to the promoter, are compared with those obtained in other systems. CAP, the catabolite activator protein of Escherichia coli, exerts a positive control on the expression of many genes, principally on those that are involved in the catabolism of substrates.In the presence of its effector, cyclic AMP (cAMP), it stimulates transcription initiation at the promoter of these genes (14,33,45a,48; de Crombrugghe et al., Biol. Reg. Dev., in press). The primary sequence of CAP is known (1, 2, 8), its three-dimensional structure has been established at 0.29-nm resolution (29), and the sequence of its binding site at several promoters has been determined. In spite of these and numerous other studies the exact mechanism whereby CAP stimulates the initiation of transcription is still a matter of debate (19,32,39,43). The most extensive studies performed so far have concerned the lac, gal, and ara operons (3, 15, 17, 20, 22, 25, 31, 40-42, 45, 47), and the results obtained in these three systems are quite different (see below). To gain more insight into the mechanism of CAP action we undertook a study of a fourth promoter, namely, the one which controls the expression of malT, the positive regulator gene of the maltose regulon (7, 1&, 11, 36). This system seemed relatively simple since the activation by CAP and cAMP is the only regulation known to be exerted on malT expression (6, 10). We determined the site at which CAP binds and demonstrated that it enhances the binding of RNA polymerase at the malT promoter and that it stimulates the initiation of transcription at this promoter, presumably by enhancing RNA polymerase binding. MATERIALS AND METHODSStrains and media. The following strains of E. coli K12 were used. pop3, which was isolated under the designation MC4100 by M. Casadaban (10), is F-araD139 Alac-169 rpsL relA thiA. pop3931 derives from pop3 and carries the malTpl mutation (6, 35). Strain JM83 (30) was supplied by P. Stragier and is ara A(lac-pro) thi rpsL [4)80 dlacZ A(lacMJ5)].Complete medium (ML) and synthetic medium (M63) were as previously described (6). Ampicillin was used at 50 ,ug/ml in solid and liquid media.Materials. Purified CAP was kindly given by B. Blazy. It was more than 98% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ...

A new target for CRP action at the malT promoter.

“…Run-off assays The assays reported above indicated that similar amounts of competent complex were formed at malT in the absence and presence of CRP. These results, obtained by monitoring the formation of a trinucleotide, were at striking variance with the single-round transcription experiments reported by Chapon and Kolb (1983), who found a significant increase in the amount of full-length transcripts synthesized when CRP was added. Run-off experiments were therefore performed under conditions analogous to the abortive initiation assays.…”

“…Organization of the DNA region upstream the RNA start at malT As already shown in Chapon and Kolb (1983), the malT promoter is extremely weak in the absence of added CRP -cAMP complex. Run-off transcription assays, performed under their conditions on the 190-bp Sall -Hinfl fragment depicted in Figure 1, yielded several faint sets of long transcripts (n > 10) ( Figure 6).…”

“…This deduction is strengthened by an analysis of the pattern of attack of this complex by DNase I or by orthophenanthroline-Cu+; protections and enhancements are weak (cf. Chapon and Kolb, 1983;A.Kolb, M.Menendez and H.Buc, unpublished).…”

“…At the malT promoter, the 5' end of the predominant message is ApUpUpA (cf. Chapon and Kolb, 1983). Open complex formation at malT was followed through the abortive synthesis of the trinucleotide ApUpU from ApU and [a-32P]UTP.…”

A common role of CRP in transcription activation: CRP acts transiently to stimulate events leading to open complex formation at a diverse set of promoters

“…Figure 2A and B shows the footprinting patterns of the coding and template strands, respectively. CRP binds to a region between -60 and -80 in the presence of cAMP as reported by Chapon and Kolb (1983). When RNAP alone was incubated with the malT DNA, a region between ϩ20 and -60 was protected against DNase I digestion.…”