Abstract
In vivo transcription of the replication region of plasmid pE194 yeidls two classes of mRNAs that encode Cop and RepF proteins, respectively. These transcripts are oriented 5' to 3' exclusively in the clockwise direction on the standard map. The cop region contains an open reading frame capable of encoding a 55-amino-acid protein that was demonstrated electrophoretically as a 6-kilodalton product synthesized in Bacillus subtilis minicells and chemically by N-terminal sequencing of a 116-kilodalton fusion protein with Escherichia coli beta-galactosidase. Four transcripts derived from the repF region were found, of which the longest, approximately 720 nucleotides, had the length, orientation, and transcription start site necessary to code for the full-length RepF protein (216 amino acid residues), deduced from the DNA sequence. The 5' ends of the shorter repF transcripts fall within the repF open reading frame. We propose that (i) cop specifies a protein rather than an RNA countertranscript, (ii) the Cop protein functions as a negative-acting element in pE194 replication by regulating synthesis of both RepF and of itself, and (iii) increased plasmid copy number can be explained in terms of cop region mutations that either reduce the intrinsic activity of Cop protein or the rate of its synthesis.