Dissecting DNA-protein and protein-protein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection.
The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.
Snapyan
Marina
M
Laboratoire de Biotechnologie, FRE-CNRS 2230 Biocatalyse, Université de Nantes, Nantes, France.
Lecocq
Michèle
M
Guével
Laetitia
L
Arnaud
Marie-Claire
MC
Ghochikyan
Anahit
A
Sakanyan
Vehary
V
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D040081:Protein Array Analysis / Q000379:methods* / Q000706:statistics & numerical data; D011485:Protein Binding; D040901:Proteomics; D012680:Sensitivity and Specificity; D019265:Spectroscopy, Near-Infrared; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins