GcvA binding site 1 in the gcvTHP promoter of Escherichia coli is required for GcvA-mediated repression but not for GcvA-mediated activation

Department of Microbiology, The University of Iowa, Iowa City, IA 52242, USA 1 Author for correspondence: George V. Stauffer. Tel: +1 319 335 7791. Fax: +1 319 335 9006. e-mail: george-stauffer{at}uiowa.edu GcvA binds to three sites in the gcvTHP control region, from base -34 to -69 (site 1), from base -214 to -241 (site 2) and from base -242 to -271 (site 3). Previous results suggested that sites 3 and 2 are required for both GcvA-dependent activation and repression of a gcvT :: lacZ fusion. However, the results were less clear as to the role of site 1. To determine the role of site 1 in regulation, single and multiple base changes were made in site 1 and tested for their ability to alter GcvA-mediated activation and GcvA/GcvR-mediated repression. Several of the mutants were also tested for effects on GcvA binding to site 1 and the ability of GcvA to bend DNA at site 1. The results are consistent with site 1 playing primarily a role in negative regulation of the gcvTHP operon. Keywords: gcvTHP , gcvT :: lacZ , GcvA, GcvR, repression Abbreviations: CRP, cyclic AMP receptor protein; GCV, glycine cleavage; RNAP, RNA polymerase; WT, wild-type