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Journal of Bacteriology, April 2002, p. 2273-2280, Vol. 184, No. 8
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.8.2273-2280.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase {alpha} Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

Nigel J. Savery,1* Georgina S. Lloyd,2 Stephen J. W. Busby,2 Mark S. Thomas,3 Richard H. Ebright,4 and Richard L. Gourse5

Department of Biochemistry, University of Bristol, Bristol,1 School of Biosciences, University of Birmingham, Birmingham,2 Division of Genomic Medicine, University of Sheffield, Sheffield, United Kingdom,3 Howard Hughes Medical Institute, Waksman Institute, and Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854 ,4 Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 537065

Received 20 September 2001/ Accepted 14 January 2002

Alanine scanning of the Escherichia coli RNA polymerase {alpha} subunit C-terminal domain ({alpha}CTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of {alpha}CTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for {alpha}CTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in {alpha}CTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

* Corresponding author. Mailing address: Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. Phone: (44) 117 928 9708. Fax: (44) 117 928 8274. E-mail: N.J.Savery{at}bristol.ac.uk.

Journal of Bacteriology, April 2002, p. 2273-2280, Vol. 184, No. 8
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.8.2273-2280.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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