AN RNA-POLYMERASE PREPARATION FROM METHYLOBACTERIUM-EXTORQUENS AM1 CAPABLE OF TRANSCRIBING FROM A METHYLOTROPHIC PROMOTER

RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM 1 cells grown on methanol or on succinate. The beta, beta', alpha and omega subunits were approximately the same size as those of Escherichi a coli, and the identity of the omega subunit was confirmed by N-termi nal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be sigma fa ctors, a 40 kDa polypeptide that shared identity with sigma(32) homolo gues, and a 97 kDa polypeptide that shared identity with sigma(70) hom ologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli sigma(70). The same complement of putative si gma factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major metha nol-inducible sigma factor is present in this strain. Run-off assays s howed that the purified RNAP was capable of initiating transcription s pecifically at the transcriptional start site of a methylotrophic gene , mxaF, which encodes the large subunit of methanol dehydrogenase and is found only in methylotrophic bacteria.