Synthesis of RNA I by the RNA polymerase from Micrococcus luteus on the Escherichia coli plasmid pBR322

Abstract

Incubation of DNA-dependent RNA polymerase from Micrococcus luteus (gram-positive) with the plasmid pBR322 under transcription conditions in vitro leads to the formation of a rather short-chained RNA. This transcript is initiated at the same site on pBR322 as RNA I, a defined Escherichia coli RNA polymerase product. M. luteus RNA polymerase initiates transcription at the RNA I site much more efficiently than the E. coli enzyme, using either a plasmid preparation of pBR322 or an appropriate linear restriction fragment as template. In the latter case, cleavage of the restriction fragment 24 or 71 nucleotides (but not 91 nucleotides) upstream of the initiation site destroys template activity. By sequence analysis it was determined that the 3' terminus of the M. luteus RNA polymerase transcription product is identical with that known for RNA I. Moreover, in agreement with synthesis of RNA I by E. coli RNA polymerase in vitro, termination by the M. luteus enzyme is also a stutter process characterized by the same dependence on the available UTP concentration. These observations lead to the hypothesis that termination by eubacterial RNA polymerases might not be species-specific.