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Transcription Activation by FNR: Evidence for a Functional Activating Region 2

The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN,¹ School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom²

Received 26 April 2002/ Accepted 29 July 2002

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2. Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters. The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted. Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the LysAla substitutions at a class II promoter. Site-directed mutagenesis of a negatively charged patch (¹⁶²EEDE¹⁶⁵) within the N-terminal domain of the RNAP subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.

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