Studies on transcription regulation of ampC β-lactamase with purified ampR

Abstract

AmpR is a trans-acting transcriptional regulator of ampC β-lactamase. As it binds to the intercistronic region of ampC-ampR genes, elucidation of its binding nature to the region will give aids to solve the transcription activation process. For this study, Citrobacter freundii AmpR was highly purified through affinity chromatography, quantified at every purification step, subjected to binding, cross-linking and kinetic studies. AmpR was purified up to 99% from DNA(1.5 kilobase-pair BamHI fragment of pNU305)-sepharose chromatography and was identified on SDS-PAGE gel. The first 15 N-terminal amino acids were in agreement with that predicted from the nucleotide sequence. Interestingly, DNA binding ability of AmpR was not dependant on purification degree. Cross-linking of purified AmpR with 0.005% glutaraldehyde shows dimer and tetramer bands on SDS-PAGE gel. On the binding studies with 236 base-pair Clal-Sacl fragment of pNU305, which carries binding sites, sigmoidal binding curve was observed suggesting that AmpR binds the DNA cooperatively. 3 protein-DNA ladders at high AmpR concentration may suggest that there would be 3 binding sites. The apparent equilibrium binding constant was calculated as 150nM. On the plot of Hill equation, the Hill coefficient and the apparent dissociation constant were calculated as 1.95 and $5 \times10^{-14}M^2$, respectively. The association and dissociation rate constants were $6.4 \times10^{10}/M^2·sec$ and $9.0 \times10^{-3}/sec$, respectively, from the binding kinetic studies. These results mean that AmpR binds very tightly to the intercistronic region of ampR - ampC genes at least at two sites, cooperatively, as dimeric or tetrameric form, with very high DNA sequence specificity. Additional protein was found to bind tightly to this region. The protein is presumed to be one of regulatory factor that can change the conformation of AmpR-DNA complex, and is needed for the transcription of ampC gene. It is reported that the trans...