In vitro determination of the effect of indoleglycerol phosphate on the interaction of purified TrpI protein with its DNA-binding sites.
Expression of the trpBA gene pair of Pseudomonas aeruginosa is regulated by the endogenous level of indoleglycerol phosphate (InGP) and the trpI gene product. The TrpI protein binds to the -77 to -32 region of the trpBA promoter. This region is divisible into two sites: site I, which is protected by TrpI in the presence and absence of InGP; and site II, which is protected by TrpI only in the presence of InGP. Recently, the trpI gene was subcloned into an expression vector and the protein was overproduced in Escherichia coli. The TrpI protein was purified to 80 to 95% purity. The molecular weight of native TrpI protein is estimated to be 129,000 by gel exclusion chromatography, and therefore it is likely a tetramer composed of 31,000-dalton monomers. Gel retardation assays with the purified TrpI protein demonstrated that InGP increases the affinity of TrpI for sites I and II approximately 17- and 14-fold, respectively. Binding of TrpI to site I is site II independent. However, the protein has low intrinsic affinity for site II and its binding to site II is site I dependent. Therefore, binding of TrpI to site II probably requires its interaction with a second TrpI molecule at site I.
Chang
M
M
Department of Microbiology, University of Iowa, Iowa City 52242.
Crawford
I P
IP
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005798:Genes, Bacterial; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators