An RNA polymerase preparation from Methylobacterium extorquens AM1 capable of transcribing from a methylotrophic promoter

Abstract

Summary: RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate. The β, β', α and ω subunits were approximately the same size as those of Escherichia coli, and the identity of the ω subunit was confirmed by N-terminal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be σ factors, a 40 kDa polypeptide that shared identity with σ32 homologues, and a 97 kDa polypeptide that shared identity with σ70 homologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli σ70. The same complement of putative σ factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible σ factor is present in this strain. Run-off assays showed that the purified RNAP was capable of initiating transcription specifically 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.