Purification and initial characterization of RNA polymerase from Thermus thermophilus strain HB8.
Utilizing a novel and rapid two-column purification procedure, the DNA-dependent RNA polymerase (RNAP) from the thermophile, Thermus thermophilus HB8, was purified to electrophoretic homogeneity with a recovery of 65% (as determined by RNAP activity) in less than 2 days. The purified enzyme was characterized using DNA containing the lambdaP(R) promoter. KMnO(4) footprinting, abortive initiation assays, and the formation of the specific stalled elongation complex provide compelling evidence that T. thermophilus RNA polymerase can bind to DNA containing the lambdaP(R) promoter, form an open complex, and initiate transcription in a temperature-dependent manner. This evidence suggests that T. thermophilus RNAP possesses less intrinsic binding energy than E. coli RNAP. Instead, T. thermophilus relies on the high temperatures of its environment to provide the thermal energy required to stimulate open promoter complex formation, initiate transcription, and facilitate the conformational changes in RNA polymerase that result in nucleotide incorporation.
Xue
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Department of Chemistry, Campus Box 3290, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
Hogan
B P
BP
Erie
D A
DA
D010582:Bacteriophage lambda / Q000201:enzymology; D002482:Cellulose / Q000031:analogs & derivatives*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D011094:Polyethyleneimine; D011196:Potassium Permanganate; D015203:Reproducibility of Results; D016963:Thermus thermophilus / Q000201:enzymology*
D011196:Potassium Permanganate; D011094:Polyethyleneimine; D002482:Cellulose; C008881:phosphocellulose; D012321:DNA-Directed RNA Polymerases