title: Analysis of interactions between Activating Region 1 of Escherichia coli FNR protein and the C-terminal domain of the RNA polymerase alpha subunit: use of alanine scanning and suppression genetics.
abstract: Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) during transcription activation at FNR-regulated promoters. Using an alphaCTD alanine scan mutant library, we have identified the residues of alphaCTD that are important for FNR-dependent transcription activation. Residues Asp-305, Gly-315, Arg-317, Leu-318 and Asp-319 are proposed to be the key residues in the contact site on alphaCTD for Activating Region 1 of FNR. In previous work, it had been shown that Activating Region 1 of FNR is a large surface-exposed patch and that the two crucial amino acid residues are Thr-118 and Ser-187. In this work, we have constructed Arg-118 FNR and Arg-187 FNR and shown that both FNR derivatives are defective in transcription activation. However, the activity of FNR carrying Arg-118 can be partially restored by substitutions of Lys-304 in alphaCTD. Similarly, the activity of FNR carrying Arg-187 can be partially restored by substitutions of Arg-317 or Leu-318 in alphaCTD. The specificity of the restoration suggests that, during transcription activation by FNR, the side-chain of residue 118 in Activating Region 1 of FNR is located close to Lys-304 and Asp-305 in alphaCTD. Similarly, the side-chain of residue 187 in Activating Region 1 of FNR is located close to Arg-317 and Leu-318 in alphaCTD. These results can be used to model the interface between Activating Region 1 of FNR and its contact target in alphaCTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcription activator, CRP and alphaCTD.
authors.affiliation: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Lee
authors.forename: H J
authors.initials: HJ
authors.lastname: Wing
authors.forename: N J
authors.initials: NJ
authors.lastname: Savery
authors.forename: S J
authors.initials: SJ
authors.lastname: Busby
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors; D001120:Arginine; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D000409:Alanine