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, have been used to investigate the molecular details of macromolecular interactions at prokaryotic promoter-operators. For the Escherichia coli methionine repressor, MetJ, we have quantitated the interaction of the protein with synthetic and natural operator sites and shown that the SPR response is directly related to the stoichiometry of the complexes being formed. The utility of a continuous flow system has also been exploited to investigate transcription from an immobilised promoter-operator fragment; with transcripts collected and subsequently characterised by RT-PCR. This technique has enabled us to investigate how repressor binding affects (i) the interaction of the RNA polymerase (RNAP) with the promoter and (ii) the ability of RNAP to initiate transcription. 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 transcription initiation to occur as soon as the repression complex dissociates. These techniques have also been used to examine protein-DNA interactions in the E. coli and Bacillus subtilis arginine repressor systems. The repressors are the products of the argR and ahrC genes, respectively. Both proteins form hexamers in rapid equilibrium with smaller subunits believed to be trimers. There are three types of operator in these systems, autoregulatory, biosynthetic and catabolic (B. subtilis only). 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.
Stockley
P G
PG
School of Biology, University of Leeds, UK. stockley@bmb.leeds.ac.uk
Baron
A J
AJ
Wild
C M
CM
Parsons
I D
ID
Miller
C M
CM
Holtham
C A
CA
Baumberg
S
S
D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014158:Transcription, Genetic
C087018:ArgR protein, Bacteria; C490177:ArgR protein, E coli; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; C060486:methionine repressor protein, Bacteria; C489220:methionine repressor protein, E coli