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

Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C-terminal domain of the RNA polymerase alpha subunit (a lpha CTD) during transcription activation at FNR-regulated promoters. Using an alpha CTD alanine scan mutant library, we have identified the residues of alpha CTD 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 alpha CTD 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 resid ues 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 transc ription activation. However, the activity of FNR carrying Arg-118 can be pa rtially restored by substitutions of Lys-304 in alpha CTD. Similarly, the a ctivity of FNR carrying Arg-187 can be partially restored by substitutions of Arg-317 or Leu-318 in alpha CTD. The specificity of the restoration sugg ests that, during transcription activation by FNR, the side-chain of residu e 118 in Activating Region 1 of FNR is located close to Lys-304 and Asp-305 in alpha CTD. Similarly, the side-chain of residue 187 in Activating Regio n 1 of FNR is located close to Arg-317 and Leu-318 in alpha CTD. These resu lts can be used to model the interface between Activating Region 1 of FNR a nd its contact target in alpha CTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcriptio n activator, CRP and alpha CTD.