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

• Published in: Microbiology (Society for General Microbiology) Vol. 146; no. 11; pp. 2909 - 2918
• Main Authors: Wonderling, Laura D, Urbanowski, Mark L, Stauffer, George V
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.11.2000; Society for General Microbiology
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Binding Sites - genetics; Biological and medical sciences; Conserved Sequence; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; GcvA protein; gcvH gene; gcvP gene; GcvR protein; gcvT gene; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetics; Microbiology; Nucleic Acid Conformation; Operon; Phenotype; Point Mutation; Promoter Regions, Genetic; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription; Operon; Escherichia coli; Transcription repressor; Binding site; Gene expression; Glycine; Metabolism; Regulation(control); Aminoacid; Bacteria; Regulatory sequence; Enterobacteriaceae
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-146-11-2909

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