Regulation of ribosomal RNA promoters with a synthetic lac operator

Abstract

A synthetic 21-base-pair long DNA fragment containing the central lac operator sequence has been inserted near the initiation point of the cloned Escherichia coli rrnB rRNA promoter P2 in the natural and reverse orientation. RNA synthesis is efficiently repressed in both orientations in lac I(q) strains and is induced with isopropyl β-D-thiogalactoside. When the rrnB promoter P1 is also present, upstream from P2 and the synthetic lac operator, repression of transcription is incomplete. The levels of transcription were measured in vivo, indirectly by the expression of a protein (chloramphenicol acetyltransferase), or directly by the expression of a stable RNA (E. coli 4.5S RNA) in a simple assay involving gel electrophoresis of unlabeled total RNA from E. coli. The rrnB promoter constructions can produce high levels of protein expression as well as high levels of expression of stable RNA.