FIG. 2.

Alanine substitutions in or near the Fis helix-turn-helix DNA binding motif. (A) Ribbon model of the crystal structure of Fis (39, 63, 77). The diagram on the left represents the Fis homodimer. The amino-terminal β-loop followed by four α-helices, α-A, α-B, α-C, and α-D, is indicated for one of the subunits. The α-B, α-C, and α-D helices of a single subunit are shown in the diagram on the right, which indicates the positions of the side chains for the residues in or near the helix-turn-helix DNA binding motif (comprised of α-C and α-D) that were replaced with alanine. (B) Generation of alanine mutations. The DNA sequence of E. coli fis is expressed as triplet codons, and the deduced amino acid sequence is shown above the DNA sequence. The positions of alanine substitutions (Ala) are indicated above the amino acid sequence, and the corresponding DNA mutations are indicated beneath the DNA sequence. The arrows indicate the regions that form β-strands (β-1 and β-2), and the boxes indicate the regions that form the four α-helices. The first and last residues of helices α-C (positions 74 and 81) and α-D (positions 85 and 94) are indicated. (C) SDS-polyacrylamide gel electrophoresis of the purified Fis proteins stained with Coomassie blue. The estimated purities of the various Fis proteins are indicated below the lanes.