DISSECTING THE MOLECULAR DETAILS OF PROKARYOTIC TRANSCRIPTIONAL CONTROL BY SURFACE-PLASMON RESONANCE - THE METHIONINE AND ARGININE REPRESSOR PROTEINS

The commercial surface plasmon resonance (SPR) biosensors, BIACORE, ha ve been used to investigate the molecular details of macromolecular in teractions at prokaryotic promoter-operators. For the Escherichia coli methionine repressor, MetJ, we have quantitated the interaction of th e protein with synthetic and natural operator sites and shown that the SPR response is directly related to the stoichiometry of the complexe s being formed. The utility of a continuous flow system has also been exploited to investigate transcription from an immobilised promoter-op erator fragment; with transcripts collected and subsequently character ised by RT-PCR. This technique has enabled us to investigate how repre ssor binding affects (i) the interaction of the RNA polymerase (RNAP) with the promoter and (ii) the ability of RNAP to initiate transcripti on. Remarkably, the repression complex appears to stabilise binding of RNAP, whilst having the expected effects on the levels of transcripts produced. This may well be a general mechanism allowing rapid transcr iption initiation to occur as soon as the repression complex dissociat es. These techniques have also been used to examine protein-DNA intera ctions in the E. coli and Bacillus subtilis arginine repressor systems . The repressors are the products of the argR and ahr-C genes, respect ively. Both proteins form hexamers in rapid equilibrium with smaller s ubunits believed to be trimers. Then are three types of operator in th ese systems, autoregulatory, biosynthetic and catabolic (B. subtilis o nly). Sensorgrams show that each protein recognises the three types of immobilised operator differently and that binding is stimulated over 100-fold by the presence of L-arginine. (C) 1998 Elsevier Science S.A. All rights reserved.