Spacing and orientation requirements of GcvA-binding sites 3 and 2 and the Lrp-binding region for gcvT::lacZ expression in Escherichia coli

• Published in: Microbiology (Society for General Microbiology) Vol. 144; no. 5; pp. 1417 - 1422
• Main Authors: Stauffer, Lorraine T, Stauffer, George V
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.05.1998; Society for General Microbiology
• Subjects: Aminomethyltransferase; Bacteriology; Bacteriophage lambda - genetics; Base Sequence; beta-Galactosidase - metabolism; Binding Sites; Biological and medical sciences; DNA, Bacterial; 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; Gene Expression Regulation, Bacterial; Genetics; Glycine - metabolism; Helix-Turn-Helix Motifs; Hydroxymethyl and Formyl Transferases - genetics; Inosine - metabolism; Lac Operon; Low Density Lipoprotein Receptor-Related Protein-1; Microbiology; Molecular Sequence Data; Operon; Promoter Regions, Genetic; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Recombinant Fusion Proteins - biosynthesis; Transcription Factors - genetics; Transcription Factors - metabolism; Operon; Transcription promoter; Escherichia coli; DNA binding protein; Binding site; Gene expression; Glycine; Regulation(control); Bacteria; Cleavage; Regulatory sequence; Cis effect; Enterobacteriaceae
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-144-5-1417

Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA ABSTRACT Both GcvA and Lrp are required for normal regulation of the gcv operon. Moving the GcvA-binding sites 3 and 2 and the Lrp-binding region either closer to, or further away from, the gcv promoter by approximately one helical turn of DNA resulted in a less than twofold decrease in glycine-mediated activation or inosine-mediated repression of a gcvT::lacZ fusion. Moving these sites approximately two helical turns of DNA away from the gcv promoter resulted in a further loss of both activation and repression; moving these sites approximately three helical turns of DNA from the gcv promoter resulted in an essentially complete loss of both glycine-mediated activation and inosinemediated repression. However, when these sites were moved by approximately 1.5 and 2.5 helical turns of DNA away from the gcv promoter, there was a complete loss of both glycine-mediated activation and inosine-mediated repression of the gcvT::lacZ fusion. The flexibility in the absolute distance of the GcvA- and Lrp-binding sites relative to the gcv promoter, but strict orientation dependence of these sites is consistent with a possible protein-protein interaction of either GcvA, Lrp, or both of these proteins with RNA polymerase. Because of the location of these target sites relative to the gcv promoter, it is also likely that DNA looping is required for this mechanism of regulation. * Author for correspondence: George V. Stauffer. Tel: + 1 319 335 7791. Fax: + 1 319 335 9006. e-mail: george-stauffer@uiowa.edu Keywords: glycine cleavage, gcvT::lacZ, GcvA protein, Lrp protein, regulation