λ po, a promoter for oop RNA synthesis, has a role in replication of plasmids derived from bacteriophage λ

Abstract

Transcription initiated at the bacteriophage λ p o promoter gives a short RNA, called oop RNA. Early studies led to a proposal that this transcript plays a role in the initiation of λ DNA replication. In fact, the p o promoter is located in the λ replication region and it was suggested that oop RNA may be a primer for replication proceeding leftward from oriλ. However, since in vitro experiments demonstrated that primers for λ DNA replication are produced by the dnaG gene product (DnaG primase) and subsequent in vivo studies indicated that oop RNA is an antisense RNA for the λ cII gene expression, the above-mentioned hypothesis has fallen into oblivion. Nevertheless, here we demonstrate that the p o promoter plays a role in λ DNA replication, indeed. We found that λ plasmids bearing a mutation that inactivates p o occur in Escherichia coli cells in a copy number significantly lower than wild-type λ plasmids. Amplification of λ p o − plasmids during the relaxed response was less efficient relative to λ p o + plasmids suggesting less frequent initiation of replication from oriλ in the absence of transcription from p o. This suggestion was confirmed by measurement of incorporation of [ 3H]thymidine into λ plasmid DNA during pulse-labeling experiments. Therefore, we propose that transcription from the p o promoter stimulates replication initiation at oriλ as suggested a long time ago, however, contrary to that suggestion, we assume that the process of p o-initiated transcription per se but not the transcription product ( oop RNA) might play a role at early steps of λ DNA replication.