Identification of the activating region of catabolite gene activator protein ( CAP ) : Isolation and characterization of mutants of CAP

We have isolated 21 mutants of catabolite gene activator protein (CAP) defective in transcription activation at the lac promoter but not defective in DNA binding. The amino acid substitutions in the mutants map to a single region of CAP: amino acids 156-162. As assessed in vito, the substituted CAP variants are nearly completely unable to activate transcription at the lac promoter but bind to DNA with the same affmity and bend DNA to the same extent as wild-type CAP. Our results establish that amino acids 156-162 are critical for transcription activation at the lac promoter but not for DNA binding and DNA bending. In the structure of CAP, amino acids 156-162 are part of a surface loop. We propose that this surface loop makes a direct protein-protein contact with RNA polymerase at the lac promoter. Escherichia coli catabolite gene activator protein (CAP; also referred to as the cAMP receptor protein, CRP) is a structurally characterized transcription activator protein (reviewed in refs. 1 and 2). CAP functions by binding, in the presence of the allosteric effector cAMP, to specific DNA sites located in or upstream of CAP-dependent promoters. The best-characterized CAP-dependent promoter is the lac promoter. The consensus DNA site for CAP is 5'-AAATGTGATCTAGATCACATTT-3' (3). The consensus DNA site is 22 bp long and exhibits perfect twofold sequence symmetry. The three-dimensional structure of CAP has been determined to 2.5 A resolution by x-ray diffraction analysis (4), and the three-dimensional structure of the CAP-DNA complex has been determined to 3.0 A resolution by x-ray diffraction analysis (5). CAP is a dimer of two identical subunits, each of which is 209 aa long. The CAP-DNA complex is twofold symmetric: one subunit ofCAP interacts with one-half of the DNA site; the other subunit of CAP interacts in a twofoldsymmetry-related fashion with the other halfofthe DNA site. CAP sharply bends DNA in the CAP-DNA complex (6, 7). The CAP-induced DNA bend is -90° in the CAP-DNA complex in the crystalline state (5) and 80°-180° in the CAP-DNA complex in solution (8-12). For a number of prokaryotic and eukaryotic transcription activator proteins, it has been possible to identify regions of the protein that are essential for transcription activation but not essential for DNA binding (reviewed in refs. 13 and 14). Such regions have been designated "activating regions" and have been proposed to participate in direct protein-protein interactions with RNA polymerase and/or basal transcription factors. An early report had suggested that CAP contains an activating region consisting, at least in part, of aa 171 of CAP 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. (15). However, that report has been shown to be incorrect (16). The objectives of the work in this report were (i) to determine whether CAP contains an activating region and, if so, (ii) to determine the location of the activating region. Our approach to these objectives was to perform random mutagenesis of the gene encoding CAP, followed by an in vivo genetic screen, in order to isolate mutants ofCAP specifically defective in transcription activation-i.e., mutants of CAP defective in transcription activation but not defective inDNA binding. We designate such mutants crpPc, where crp denotes the gene encoding CAP, and pc denotes defective in positive control of transcription. MATERIALS AND METHODS Strains and Plasmids. Strains XE64.1 (Acrp39 strA thi) and XE82 (Acrp39 strA thi lacPUVS-OCAP) are described in ref. 16. Plasmid pYZCRP is described in ref. 17. Plasmid pAGICAP was constructed by insertion of a 39-bp DNA sequence containing the consensus DNA site for CAP between the Xba I and Sal I sites of plasmid pBend3 (kindly supplied by S. Adhya; cf. ref. 10). The insertion was made by use of site-directed mutagenesis [method of ref. 18, except that template DNA was prepared in E. coli strain XL1-Blue (Stratagene)]. The primer used was 5'-GGGATCCTCTAGAGGGTGCCTAAAATGTGATCTAGATCACATTTATTGCGTTGTCGACACGCGTAGATC-3'. Mutagenesis. Random mutagenesis of the crp gene of plasmid pYZCRP was performed by use of PCR with Taq DNA polymerase as described in ref. 17. The primers used were 5'-CAGTCAGGATGCTACAGTAATACA-3' and 5'ATCTTCCCCATCGGTGATGTCGGC-3'. Purification of CAP and CAP Derivatives. CAP and CAP derivatives were purified by cAMP affinity chromatography as described in ref. 19. The fraction of molecules active in sequence-specific DNA binding (0.3-0.8 for CAP, [Ala158]CAP, [Ile-158]CAP, [Leu-159]CAP, and [Arg-159]CAP; 0.05 for [Cys-162]CAP) was determined by titration ofa 40-bp DNA fragment having the consensus DNA site for CAP under stoichiometric binding conditions (3). Transcription Experiments. Abortive initiation in vitro transcription experiments were performed as described in ref. 16. Experiments were performed using as templates a 203-bp EcoRI/EcoRI DNA fragment of plasmid pBR-203-lac and a 314-bp Ava II/EcoRI DNA fragment of bacteriophage M13mp2 ICAP. Experiments with CAP and [Ala-158]CAP also were performed using as templates a 203-bp EcoRI/ EcoRI DNA fragment of plasmid pBR-203-lacPUV5 and a Abbreviation: CAP, catabolite gene activator protein. *To whom reprint requests should be addressed at: Waksman Institute, Rutgers University, New Brunswick, NJ 08855-0759.