Mutants of the catabolite activator protein of Escherichia coil that are specifically deficient in the gene-activation function ( 1 acZ fusions / transcription in vitro / protein-protein interaction / RNA polymerase )

In the presence of cyclic AMP, the catabolite activator protein (CAP) of Escherichia coli binds DNA and stimulates transcription at a number of promoters. We have examined a model ofCAP bound at the gal promoter and, using directed mutagenesis, have isolated CAP mutants that are analogous to the X repressor positive control (pc) mutants. These CAP mutants bind DNA but are defective in stimulating transcription at the gal PI promoter. These mutants are also altered in positive control at the lac and malT promoters, where CAP binds to sites further upstream from the transcription start site. The catabolite activator protein (CAP; also known as cyclic AMP receptor protein, CRP) is both a positive and a negative regulator of gene transcription. In the presence of cyclic AMP, CAP dimers bind to 22-base-pair sites of related DNA sequence near certain promoters (1-4). The distance between the known CAP binding site and the transcription start site at different promoters varies. At the gal P1 promoter, CAP stimulates transcription by binding to a site centered at about position -42 relative to the transcription start point (position + 1). In contrast, at lac the identified CAP site is centered at about -60, and at malT it is centered at about -70 (Fig. 1). How does CAP stimulate transcription when bound at these various distances from the transcription start sites? We believe that X repressor bound to DNA activates transcription by contacting RNA polymerase and helping it to form an open complex at the adjacent promoter (5, 6). Isolation of X repressor mutants that bind DNA normally, or nearly normally, but fail to stimulate transcription (6, 7) supports this model. These positive control (pc) mutants bear altered amino acid residues that lie on a surface of X repressor predicted from the crystal structure, and other information, to be near polymerase at the adjacent promoter (6). In this paper we describe the isolation of pc mutants of CAP. From the known crystal structure of CAP (8) and the inferred structure of the CAP-DNA complex, we predict the surface ofCAP that would most likely contact polymerase at the gal P1 promoter. We used site-directed mutagenesis to change residues on or near this surface of CAP and, as a control, residues on another surface that we predict would not contact polymerase. Here we report the isolation of three CAP mutants bearing changed residues on or near the designated surface. Each mutant manifests the pc phenotype-that is, they bind to DNA but fail to stimulate fully transcription at gal promoter P1. Intriguingly, these mutants also alter positive control at promoters where CAP binds at different positions relative to the start point of transcription. MATERIALS AND METHODS Enzymes and Reagents. Escherichia coli RNA polymerase; the restriction endonucleases EcoRV, HindIII, EcoRI, BamHI, and Pvu II; the Klenow fragment of E. coli DNA polymerase I; and polynucleotide kinase were purchased from New England Biolabs. [a-32P]CTP and [a-32P]dATP were obtained from New England Nuclear. Bacterial Strains. The bacterial strains used were E. coli N11000 (Acrp39, rpsL, thi), NI1001 {Acrp39, rpsL, thi, AlacU169, relA, srl: :TnJO mal7250 [I(malT-lacZ)542-J (hyb)maltp7]}, N11002 (Acrp39, rpsL, thi, AlacU169, relA, srl:: TnJ0 galE-lacZ), and KC1071 (NI1000 with the lac promoter region converted to a lacUV5 promoter and the lac operator mutated to a symmetric CAP binding site). Plasmids. pNI110 contains the HindIII-BamHI fragment carrying crp (the gene encoding CAP) from the M13 phage used for mutagenesis and the BamHI-Pvu II fragment from plasmid pBR322 cloned into HindIII/Pvu II-digested ptacl2 (9). pNI55 is pNI110 carrying the Glu-55--Lys mutation. pNI191 is pNIllO carrying the Glu-191-*Lys mutation. pKC111 is pHA5 (10) deleted for the HindIII fragment upstream of crp. pKC112 was derived from pKC111 by deletion of the HindIII fragment carrying the crp gene. pNI111 carries a HindIII-BamHI fragment from the M13 phage used for mutagenesis encoding the Glu-171--*Lys mutant crp cloned into HindIII/BamHI-digested pKC111. pNI112 is the same as pNI111 except that it carries the Glu-171-->Gln mutation. pRW75 carries the lac promoter. pNI10 is pRW75 with a wild-type crp HindIII-BamHI fragment cloned downstream of the lac promoter. pNI4 is the same as pNI10 except that it carries the Gln-170-(Lys mutation. Mutagenesis and Sequencing. Site-directed mutagenesis of the crp gene was performed essentially as described by Zoller and Smith (11). The single-stranded templates used for primer extension were prepared from M13mp19 carrying the HindIII-EcoRV fragment encoding crp from pHA5. Synthetic primers containing a single mismatch were extended in vitro with the Klenow fragment of E. coli polymerase I, and the DNA was used to transform E. coli JM101 (from J. Messing, Waksman Institute and Rutgers University, New Brunswick, NJ). Purified plaques were screened by DNA sequencing by the method of Sanger et al. (12). The DNA sequence of the entire coding region of crp was determined. The HindIII-BamHI fragment encoding CAP was cloned into expression vectors. Gel Binding Assays. A 240-base-pair fragment containing the lac CAP site was obtained by digestion of pKC120 with Pvu II and either Hind1II or EcoRI. Each fragment was end-labeled by use of Klenow fragment (13). Culture extracts were prepared as described by Brunelle et al. (14). Standard Abbreviation: CAP, catabolite activator protein. 8315 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8316 Biochemistry: Irwin and Ptashne