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

Abstract

GcvA binds to three sites in the gcvTHP control region, from base N34 to N69 (site 1), from base N214 to N241 (site 2) and from base N242 to N271 (site 3).Previous results suggested that sites 3 and 2 are required for both GcvAdependent 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.

Glycine binds the transcriptional accessory protein GcvR to disrupt a GcvA/GcvR interaction and allow GcvA-mediated activation of the Escherichia coli gcvTHP operon

“…Plasmids pGS420 and pGS428 were derived from pGS318 and carry a 15 bp and a 25 bp insert between GcvA binding site 3j2 and site 1, respectively (Stauffer & Stauffer, 1998b). Plasmid pGS357 has a 4 bp change in site 1 that reduces GcvA binding to site 1 about twofold (Wilson et al, 1995 ;Wonderling et al, 2000). These plasmids were used as the starting templates in PCR reactions with primers containing XbaI sites and sequences complementary to gcv upstream and downstream the GcvA binding sites.…”

“…1). All three of these sites are required for repression of the operon, while only sites 2 and 3 appear to be necessary for activation (Wilson et al, 1995 ;Wonderling et al, 2000). In addition, DNase I footprint studies with GcvA showed hypersensitive cleavage sites in the region between the upstream sites 3j2 and the downstream site 1 when GcvA was bound to these sites.…”

“…possible. We also used a DNA fragment carrying the three GcvA binding sites but with a 4 bp change in site 1 that reduced the affinity of GcvA for site 1 about twofold and that resulted in a loss of GcvA-mediated repression (Wilson et al, 1995 ;Wonderling et al, 2000). The ability of GcvA to bind sites 2j3 and bend the DNA were unchanged (Fig.…”

“…ducted using glycine-buffered gel electrophoresis systems (Jourdan & Stauffer, 1999 ;Wonderling et al, 2000 ;Wilson et al, 1995). Since glycine acts as the coinducer with this LTTR controlled system, effects of the small molecule on the ability of GcvA to bind its specific sites in either the gcv or gcvA control region, or to mediate bending of the DNA when bound to these sites could not be examined under the assay conditions used.…”

“…Since GcvR has no repressor capabilities in the absence of GcvA (Ghrist & Stauffer, 1995), the results suggest that a GcvR\GcvA interaction might be required for repression. Furthermore, since mutations in any of the three GcvA binding sites result in reduced repression (Wilson et al, 1995 ;Wonderling et al, 2000), at least part of the repression response of the gcv operon likely requires that a GcvR\GcvA repressor complex be bound to gcv DNA.…”