A Rho-dependent transcription termination site regulated by bacteriophage P4 RNA immunity factor

Abstract

The genes required for replication of the temperate bacteriophage P4, which are coded by the phage left operon, are expressed from a constitutive promoter (PLE). In the lysogenic state, repression of the P4 replication genes is achieved by premature transcription termination. The leader region of the left operon encodes all the genetic determinants required for prophage immunity, namely: (i) the P4 immunity factor, a short, stable RNA (CI RNA) that is generated by processing of the leader transcript; (ii) two specific target sequences that exhibit complementarity with the CI RNA. RNA-RNA interactions between the CI RNA and the target sites on the mRNA leader region are essential for transcription termination. To understand how transcription termination is elicited by the P4 immunity mechanism, it is relevant to identify the transcription termination site. This, however, could not be directly inferred from the 3'-end of the transcription products because of the extensive and complex processing and degradation of the leader RNA. In this work, by making use of a tRNA gene as a reporter, we identify the termination site of the immunity transcripts (timm). This is a Rho-dependent terminator located within the first translated gene of the left operon and is regulated by P4 immunity. Analysis of the P4 transcription pattern in Escherichia coli rho mutants suggests that termination at timm may also be important for the efficient processing of the CI RNA.