In vitro binding of purified NahR regulatory protein with promoter Psal.
The NahR regulatory protein activates the naphthalene catabolic operon through binding to the Psal promoter in the presence of salicylate. Here, we investigated in vitro binding interaction between NahR and Psal using purified functional recombinant NahR. The T7-tagged NahR was shown to exist as a monomer in solution. Electrophoretic mobility shift assay (EMSA) showed that purified NahR bound to Psal in 3 different forms, whereas surface plasmon resonance (SPR) showed on an SPR chip at ratios ranging from 1:1 (at 0.42 microM NahR) to 8:1 (at 6.8 microM NahR). The binding was slightly inhibited by salicylate, suggesting that salicylate may not be involved in the binding of NahR to the promoter, but rather may be important in the activation of prebound NahR. An examination of the binding kinetics by SPR for the interaction between NahR and Psal revealed that the equilibrium dissociation constant was approximately 2.44 x 10(-6) M and the association and dissociation rates were 7.82 x 10(4) M(-1) s(-1) and 0.191 s(-1), respectively. These results demonstrate for the first time that purified NahR binds as a monomer to Psal and undergoes multimerization. In addition, we present novel data on the kinetics of NahR binding.
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan, 609-735, Republic of Korea.
Hoo Hwi
HH
Park
Woon Ki
WK
Lim
Hae Ja
HJ
Shin
D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry*; D001665:Binding Sites; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000032:analysis / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012459:Salicylates / Q000737:chemistry*; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry*
D001426:Bacterial Proteins; C063173:NahR protein, Bacteria; D011994:Recombinant Proteins; D012459:Salicylates; D014157:Transcription Factors; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase