Characterization of activating region 3 from Escherichia coli FNR

Transcription activation of anaerobically induced genes in Escherichia coli is mediated through the action of the global anaerobic regulator FNR. Although regions of FNR involved in FNR-dependent transcription activation have been identified, the side-chains critical to the function of these regions are not known. In this study, alanine-scanning of amino acid residues 80-89 of FNR-activating region 3 (FNR-AR3) was used to determine which amino acid side-chains are required for transcription activation of class II FNR-dependent promoters. In vivo β-galactosidase assays and in vitro transcription activation assays showed that Ala substitution of Ile81, Gly85 and Asp86 had the largest transcription activation defects, while comparison of the activity of single and double mutants indicated that Thr82, Glu83, Glu87 and Gln88 may contribute in a minor way to FNR-AR3 function. Site-directed mutagenesis of positions 81 and 86 showed that the hydrophobicity of Ile81 and the negative charge of Asp86 were important to FNR-AR3’s function. Lastly, substitution of residues of E. coli FNR-AR3 with those more basic residues found in a subset of FNR homologs, such as Rhodobacter sphaeroides FnrL, resulted in a mutant strain that was unable to activate transcription from E. coli class II FNR-dependent promoters. In conclusion, this study demonstrates a requirement for negatively charged and hydrophobic side-chain residues in E. coli FNR-AR3 function, although there is likely to be some variability in the characteristics of this region in other members of the FNR family.