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

Juan Davagnino^1,

Department of Chemical Engineering, Box 357242, University of Washington, Seattle, WA 98195-1750, USA
Box 351750 and Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195-1750, USA

ABSTRACT

Summary: RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate. The β, β', and subunits were approximately the same size as those of Escherichia coli, and the identity of the subunit was confirmed by N-terminal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be factors, a 40 kDa polypeptide that shared identity with ³² homologues, and a 97 kDa polypeptide that shared identity with ⁷⁰ homologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli ⁷⁰. The same complement of putative factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible 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.

Author for correspondence: Mary E. Lidstrom. Tel: +1 206 616 5282. Fax: + 1 206 616 5721. e-mail: lidstrom@u.washington.edu

Present address: Baxter Healthcare, Co., 1720 Flower Avenue, Duarte, CA 91010, USA.