In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms

Abstract

Analysis of Clostridium pasteurianum genomic DNA indicates that the ferredoxin (Fd) gene is present in a single copy. The cloned Fd gene previously described (Graves, M.C., Mullenbach, G. T., and Rabinowitz, J. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1653-1657) was used to map in vivo and in vitro synthesized Fd transcripts. The in vivo mRNA was sized in two ways: by Northern hybridization analysis, and more directly from the known DNA sequence after the 5'- and 3'-termini were identified. The 5'-end was determined by primer extension-dideoxy sequencing and the 3'-end by S1 nuclease mapping. The monocistronic Fd mRNA contains about 255 nucleotides and, thus, is one of the shortest bacterial mRNAs yet described. We also examined the Fd transcripts produced by Escherichia coli transformed with the plasmid containing the Fd gene. E. coli RNA polymerase most likely recognizes the same promoter (P1) as the clostridial polymerase, and furthermore, efficiently uses an additional promoter (P2) that is poorly recognized by the normal host enzyme. For comparison, in vitro transcripts were generated by E. coli and Bacillus subtilis RNA polymerases. In vitro, only promoter P1 is used by either E. coli or B. subtilis RNA polymerase. The 3'-end of each of the four types of transcripts occurs essentially at the same location and maps to within a large dyad symmetry element. Comparison of the Fd promoter with other Gram-positive promoters reveals that some sequences outside of the traditional Pribnow and -35 regions are conserved. This analysis indicates that an "extended" promoter recognition site may be required in these organisms.