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

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 of T4 DNA as templates; however, it is two 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 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 adenosine ribonucleoside triphosphate. 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 six to ten 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.