Surface plasmon resonance for measuring TBP-promoter interaction.
A procedure was developed for real-time measurement of the interaction between an archaeal TATA-binding protein (TBP) with stress-gene promoters from the archaeon Methanosarcina mazeii using surface plasmon resonance (SPR), the BIACORE 3000 equipment, and the SA (streptavidin) Sensor Chip. Measurements were based on the SPR optical phenomenon, which resulted in light extinction when TBP bound a promoter. This process, detected as a change in a particular angle, was recorded in a sensorgram. The BIA-evaluation program allowed the calculation of the equilibrium constant (K(A)) of the interaction of M. mazeii TBP with the promoters of the stress genes grpE, hsp70(dnaK), and hsp40(dnaJ) (0.47, 0.26, and 1.21x10(7)M(-1), respectively) and, for comparison, with the promoter of a non-heat-shock gene, orf16 (0.08x10(7)M(-1)). The association rate (k(a)) of the non-heat-shock gene orf16 was 0.4x10(4)M(-1)s(-1) and those for the stress genes, grpE, hsp70(dnaK), and hsp40(dnaJ) were higher: 2.8, 1.5, and 3.5x10(4)M(-1)s(-1), respectively. The new procedure will allow a comparative analysis of different TPBs and promoters (wild type and mutants) under physiologic and stress conditions, and a correlation of TBP binding parameters with constitutive and stress-induced gene expression.
Conway de Macario
Everly
E
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201-0509, USA. everlym@wadsworth.org
Rudofsky
Ulrich H
UH
Macario
Alberto J L
AJ
D019843:Archaeal Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D019766:Genes, Archaeal; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics; D017020:Methanosarcina / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D035181:TATA-Box Binding Protein / Q000378:metabolism*
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D019641:DNA, Archaeal; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D035181:TATA-Box Binding Protein; C063842:dnaK protein, E coli