Purification and initial characterization of RNA polymerase from Thermus thermophilus strain HB8

Utilizing a novel and rapid two-column purification procedure, the DNA-depe ndent RNA polymerase (RNAP) from the thermophile, Thermus thermophilus HB8, was purified to electrophoretic homogeneity with a recovery of 65% (as det ermined by RNAP activity) in less than 2 days. The purified enzyme was char acterized using PNA containing the lambdaP(R) promoter. KMnO4 footprinting, abortive initiation assays, and the formation of the specific stalled elon gation complex provide compelling evidence that T. thermophilus RNA polymer ase can bind to DNA containing the lambdaP(R) promoter, form an open comple x, and initiate transcription in a temperature-dependent manner. This evide nce suggests that T. thermophilus RNAP possesses less intrinsic binding ene rgy than E. coli RNAP. Instead, T. thermophilus relies on the high temperat ures of its environment to provide the thermal energy required to stimulate open promoter complex formation, initiate transcription, and facilitate th e conformational changes in RNA polymerase that result in nucleotide incorp oration.