Cro repressors from engineered mutagenesis of a synthetic cro gene ( DNA synthesis / synthetic gene / protein - DNA interactions )

A portion of the gene coding for the Cro repressor protein of bacteriophage X has been chemically synthesized, incorporating base pair changes that generate restriction endonuclease sites without altering the amino acid coding sequence. These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations. Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding to X operator OR3 in vivo. Mutations directed into the region of the cro gene encoding the a-3 helix produced altered Cro proteins with a range of affinities for operator DNA. These changes suggest which amino acids play an important role in Cro-0R3 complex formation. Recently, several new lines of investigation have provided substantial insight into the mechanism by which sequencespecific DNA-binding proteins recognize and bind to sites on double-stranded DNA. Of fundamental importance has been the elucidation of the structures of three DNA-binding proteins: the catabolite gene activator protein (CAP) from Escherichia coli (1), the amino-terminal fragment of cI repressor protein from bacteriophage X (2), and Cro repressor protein, also from X (3). All three proteins show structural similarities, the most notable of which is a protruding bihelical unit. It has been proposed that in each case one of the helices provides most of the sequence-specific contacts within the major groove of B-DNA (4-6). Furthermore, sequence homologies among other DNA-binding proteins suggest that this two-helix motif may be a common feature of DNA recognition (6-8). Cro protein, the smallest repressor characterized to date, binds as a dimer to six sites of 17 base pairs each, which are clustered into two operator regions on the phage genome (9). Refinement of the structure of Cro at high resolution has allowed a detailed model to be proposed for complex formation with operator DNA (10). The model predicts several specific contacts between each Cro monomer and base pairs in the major groove. These contacts involve amino acid side chains either within or near the a-3 helix of Cro. In addition, several sequence-independent interactions between the protein and the DNA backbone have been predicted (10). In this report we describe the design, construction, and cloning of a synthetic cro gene fragment, which allow specific mutations in the coding region to be made with ease. The effect of several such mutations on Cro repressor binding to operator OR3 is also described. The results support the current model for the interaction of Cro repressor with operator DNA. NOBLE*t, LAURENT P. BRACCO*, DAVID R. DODDS*, MATERIALS AND METHODS Bacterial Strains, Bacteriophage, and Plasmids. E. coli strain 71-18 (Mlac-pro/F' laCjq ZAM15 pro') was lysogenized with the temperate bacteriophage X112A265prmup-1, which contains the immunity region from phage 21 and a P,,n-lacZ operon fusion (11). In addition, it bears a deletion of OR1 (A265) and carries a mutation (prmup-1) that increases the level of transcription initiated at Prm in the absence of repressor. This lysogen was transformed with the plasmids pTR214, pTR190, or the pTR190 derivatives constructed in this work. Plasmids pTR214 and pTR190 contain the lac UV5 promoter operator region fused to cro (12). Molecular Cloning. Isolation of plasmid, DNA cleavage with restriction endonucleases, isolation ofDNA fragments, ligation with T4 DNA ligase (Bethesda Research Laboratories), and transformation of E. coli were carried out as de-