DNA-DEPENDENT RNA-POLYMERASE FROM ENTEROBACTER-CLOACAE IS CLOSELY-RELATED TO ESCHERICHIA-COLI

The RNA polymerase holoenzyme (RNAP) of Enterobacter cloacae was purif ied by gel filtration and heparin affinity chromatography and shown to consist of four subunits (beta', beta, alpha and sigma) of 156, 151, 45 and 82 kDa, as measured by SDS-PAGE, The 82 kDa protein was shown t o be related to the Escherichia coli primary sigma factor by western b lot analysis with polyclonal antisera raised against purified E. coli sigma(70). Functional reconstitution of E. cloacae core enzyme with pu rified E. coli sigma(70) showed that E, cloacae and E. coli sigma fact ors are closely related, The RNAP of E, cloacae initiated transcriptio n from the tac promoter with an efficiency similar to that of E. coli, Measuring promoter-specific transcription, the dependence of holoenzy me activity on salt, divalent cation and temperature was also similar to that of the E. coli RNAP, We also showed that the transcriptional i nhibitor, rifampicin, inhibits the enzyme activity of the purified RNA P of E. cloacae and E, coli at similar concentrations, We conclude, ba sed on these data, that the RNAP of E, cloacae and E. coli, both enter obacteria, are closely related, (C) 1997 Published by Elsevier Science Ltd. All rights reserved.