Purification of RNA polymerase and transcription‐termination factor Rho from Erwinia carotovora

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Abstract
Erwinia carotovora RNA polymerase consists of the holoenzyme structure .sigma.2.beta..beta.''.sigma. as found in Escherichia coli and other bacteria. E. carotovora RNA polymerase can synthesize RNA using .lambda., T7 or T4 DNA as templates; however, it is 2 times less active on these templates and is more temperature-sensitive than the E. coli enzyme. The .alpha. subunit of the E. carotovora enzyme is lower in molecular mass than its E. coli counterpart. The .sigma. factors from E. coli and E. carotovora are similar in size and in their ability to stimulate RNA synthesis by core enzyme on DNA templates such as T7 DNA. An additional protein of 115,000 Da[dalton] molecular mass, termed .gamma., is found associated with E. carotovora RNA polymerase. The .gamma. protein is tightly associated with the polymerase subunits as it is not dissociated by gel filtration in buffer containing 0.5 M NaCl. It can be purified by passing the Agarose 1.5 m enzyme through coupled Bio-Rex 70 and DEAE-cellulose columns. The .gamma.-protein, when present in excess over the .sigma. subunit, inhibits holoenzyme activity on T7 DNA but not on poly[d(A-T)] and may thus interfere with .sigma. activity. The .gamma. protein by itself cannot transcribe T7 DNA or poly[d(A-T)], nor does it stimulate core enzyme activity on T7 DNA. E. carotovora rho has a subunit molecular mass of 48,000 Da and exhibits RNA-dependent phosphohydrolysis of ATP. E. coli and E. carotovora .rho. are indistinguishable immunologically, as total fusion of precipitin bands is observed. E. carotovora .rho. elutes from a phosphocellulose column at a salt concentration of about 0.21 M KCl, compared to that of 0.29 M KCl for E. coli .rho.. The poly(C)-dependent ATPase activity of E. carotovora .rho. is more temperature sensitive and is 6-10 times less active than that of E. coli .rho.. E. carotovora .rho. is capable of terminating RNA transcripts, as indicated by a decrease in RNA synthesis using .lambda. or T7 DNA as template and E. carotovora or E. coli polymerase as the transcribing enzyme.