Structural analysis of the transcriptional activation region on Fis: Crystal structures of six Fis mutants with different activation properties

The Fis protein regulates gene expression in Escherichia coli by activating or repressing transcription of a variety of genes. Fis can activate transc ription when bound to DNA upstream of the RNA-polymerase-binding site, such as in the rrnB P1 promoter, or when bound to a site overlapping the -35 RN A polymerase binding site, such as in the proP P2 promoter. It has been sug gested that transcriptional activation in both promoters results from inter actions between specific amino acids within a turn connecting the B and C h elices (the BC turn) in Fis and the C-terminal domain of the alpha-subunit of RNA polymerase (alpha CTD of RNAP). Here, crystal structures of six Fis BC turn mutants with different transcriptional activation properties, Q68A, R71Y, R71L, G72A, G72D and Q74A, were determined at 1.9 to 2.8 Angstrom re solution. Two Of these mutants, R71Y and R71L, crystallized in unit cells w hich are different from that of wild-type Fis, and the structure of R71L of fers the most complete Fis model to date in that the extended structure of the N-terminal region is revealed. The BC turn in all of these mutant struc tures remains in a nearly identical gamma gamma beta-turn conformation as p resent in wild-type Fis. Analyses of the molecular surfaces of the transact ivation region of the mutants suggest that several residues in or near the BC turn, including Gln68, Arg71, Gly72 and Gln74, form a ridge that could c ontact the alpha CTD of RNAP on one side. The structures and biochemical pr operties of the mutants suggest that Arg71 is the most critical residue for contacting RNAP within this ridge and that the glycine at position 72 help s to stabilize the structure. (C) 2000 Academic Press.