The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription

Abstract

The lactose (lac) operon promoter is positively regulated by the catabolite gene activator-cyclic AMP complex (CAP) that binds to the DNA located 61.5 bp upstream of the transcription start site. Between the CAP binding site and the core promoter sequence is a 13-bp sequence (from ؊38 to ؊50 [the ؊45 region]). The possible roles of the ؊45 region in determining the CAP-independent level of lac expression and in the CAP activation process were studied by isolating and characterizing random multisite mutations. Only a small percentage of mutants have dramatic effects on lac promoter activity. Among the mutations that did affect expression, a 26-fold range in lac promoter activity in vivo was observed in the CAP-independent activity. The highest level of CAP-independent lac expression (13-fold the level of the wild-type lac promoter) correlated with changes in the ؊40 to ؊45 sequence and required an intact RNA polymerase ␣ subunit for in vitro expression, as expected for an upstream DNA recognition element. Mutant promoters varied in their ability to be stimulated by CAP in vivo, with levels ranging from 2-fold to the wild-type level of 22-fold. Only a change of twofold in responsiveness to CAP could be attributed to direct DNA sequence effects. The ؊40 to ؊45 sequence-dependent enhancement of promoter activity and CAP stimulation of promoter activity did not act additively. The mutant promoters also displayed other characteristics, such as the activation of nascent promoter-like activities overlapping lac P1 and, in one case, replicon-dependent changes in promoter activity.Transcription is a multistep process that begins with the binding of RNA polymerase (RNAP) to specific DNA sites termed promoters. A majority of Escherichia coli promoters (those recognized by the 70 holoenzyme) exhibit a general structural pattern consisting of two conserved hexameric sequences separated by approximately 17 bp, termed the core promoter. The strength of RNAP-core promoter binding, often correlated with the level of transcription, is determined by a number of factors. The major factors include the match of the promoter hexamers and DNA spacer to the established consensus sequence (9), the presence of activator binding sites (1) and functional activator proteins, and the presence of an upstream DNA recognition element (called an UP element) in some strong promoters, such as rrnB P1 and rrnB P2 (25; for a review, see reference 23). The UP element is recognized by the carboxyl-terminal domain of the RNAP ␣ subunit (␣-CTD).E. coli lac P1 is a well-studied 70 promoter containing a relatively weak core promoter. In addition, the sequence immediately upstream of the core promoter (Ϫ38 to Ϫ50, hereinafter called the Ϫ45 region) is a poor match to the sequences of UP elements frequently associated with highly active promoters (25). lac P1 is highly regulated, being subject to both repression and activation. Repression is maintained by a functional lacI gene product, coincident with the absence of a suitable inducer. Activation is achieved...

Sigma70 Promoters in Escherichia coli: Specific Transcription in Dense Regions of Overlapping Promoter-like Signals

“…Results of applying Cover in the region of the lac promoter, and the promoter-like signals detected experimentally in this region. P1, P2, P3, P-2A, and P þ 1T corresponding to Figure 1 presented by Reznikoff,38 and P4 to Figure 1 presented by Czarniecki et al 45 Four of five Promoter by Cover (PC) predictions form a cluster similar to that seen experimentally. PC2 corresponds to P1, the functional lac promoter.…”

Cyclic AMP Receptor Protein Regulates cspE , an Early Cold-Inducible Gene, in Escherichia coli

“…Further, the 265 determinant of ␣-CTD, which is important for ␣-CTD-UP element interaction, also was shown to contribute to P cspE transcription. The A/T-rich sequence just upstream of the Ϫ35 element (GTAAAAGTTA) in P cspE could act as a potential UP element, contributing to transcription in a CRPindependent manner, as seen for P rrnB (37) and P lac (10). In line with this, the R265A substitution in ␣-CTD caused a 50% decrease in P cspE activity in the presence of the plasmid control (pLG339 with no crp) (data not shown), indicating that the R265 residue indeed contributes to CRP-independent transcription from P cspE .…”

Genome-wide Functional Characterization of Escherichia coli Promoters and Sequence Elements Encoding Their Regulation

“…We first examined the lacZYA promoter, a classic gene regulation model whose sequence motifs are well characterized. This promoter is known to contain a variety of regulatory motifs, including twin LacI repressor sites centered at +11 and -82 (Flashner and Gralla, 1988) , a CAP activator site centered at -61 (Czarniecki, Noel and Reznikoff, 1997) , and a σ70 RNAP binding site. Our analysis revealed distinct signals corresponding to each of these sites, as well as quantitative measurements for their contribution to expression ( Figure 5C ).…”