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Surface plasmon resonance for measuring TBP-promoter interaction.

Abstract：

: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.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Conway de Macario E,Rudofsky UH,Macario AJ

doi

10.1016/s0006-291x(02)02388-4

subject

Has Abstract

pub_date

2002-11-15 00:00:00

pages

625-31

issue

5

eissn

0006-291X

issn

1090-2104

pii

S0006291X02023884

journal_volume

298

pub_type

杂志文章

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