Sequence specific interaction between promoter DNA and Escherichia coli RNA polymerase: comparative thermodynamic analysis with one immobilized partner.
Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage DeltaD(III) T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an excellent substrate for Ni(II) ions, while the SPR studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/-12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/-3.6) kcal mol(-1). The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/-2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India.
Abantika
A
Ganguly
Priya
P
Rajdev
Dipankar
D
Chatterji
D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D055767:Immobilized Proteins / Q000378:metabolism; D007700:Kinetics; D018625:Microscopy, Atomic Force; D009532:Nickel / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D055767:Immobilized Proteins; D009532:Nickel; D004247:DNA; D012321:DNA-Directed RNA Polymerases