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Journal of Bacteriology, March 2005, p. 1724-1731, Vol. 187, No. 5
0021-9193/05/$08.00+0     doi:10.1128/JB.187.5.1724-1731.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Additional Determinants within Escherichia coli FNR Activating Region 1 and RNA Polymerase Subunit Required for Transcription Activation

Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin¹

Received 26 August 2004/ Accepted 19 November 2004

The global anaerobic regulator FNR is a DNA binding protein that activates transcription of genes required for anaerobic metabolism in Escherichia coli through interactions with RNA polymerase (RNAP). Alanine-scanning mutagenesis of FNR amino acid residues 181 to 193 of FNR was utilized to determine which amino acid side chains are required for transcription of both class II and class I promoters. In vivo assays of FNR function demonstrated that a core of residues (F181, R184, S187, and R189) was required for efficient activation of class II promoters, while at a class I promoter, FF(–61.5), only S187 and R189 were critical for FNR activation. Site-directed mutagenesis of positions 184, 187, and 189 revealed that the positive charge contributes to the function of the side chain at positions 184 and 189 while the serine hydroxyl is critical for the function of position 187. Subsequent analysis of the carboxy-terminal domain of the subunit (CTD) of RNAP, using an alanine library in single copy, revealed that in addition to previously characterized side chains (D305, R317, and L318), E286 and E288 contributed to FNR activation of both class II and class I promoters, suggesting that CTD region 285 to 288 also participates in activation by FNR. In conclusion, this study demonstrates that multiple side chains within region 181 to 192 are required for FNR activation and the surface of CTD required for FNR activation is more extensive than previously observed.

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