Sequence and deletion analysis of the recombination enhancement gene (ref) of bacteriophage P1: evidence for promoter-operator and attenuator-antiterminator control

Abstract

The ref gene of bacteriophage P1 stimulates recombination between two defective lacZ genes in the Escherichia coli chromosome (lac x lac recombination) and certain other RecA-dependent recombination processes. We determined the DNA sequence of the 5' portion of the ref gene and tested various regions for functionality by inserting DNA fragments lacking increasing amounts of 5' sequence into plasmid and lambda phage vectors and measuring the ability of the constructs to stimulate lac x lac recombination. The region found essential for Ref activity in the absence of external heterologous promoters encodes two presumptive promoters, pref-1 and pref-2, whose -10 regions fall in a nearly perfect 13-base-pair (bp) tandem repeat. The -10 region of the putative pref-1 is part of a phage P1 c1 repressor recognition sequence. The first two ATG codons in the ref reading frame are, respectively, 90 and 216 bp downstream from the putative promoter-operator region. Deletion analysis indicated that translation can initiate at either ATG (although neither is associated with a canonical ribosome-binding sequence) and that the 42 amino acids in between are not indispensable for Ref stimulation of lac x lac recombination. However, the shorter reading frame appears to encode a less active polypeptide. The 91-bp leader region between the putative promoter-operator and the first ATG contains 30 codons in frame with the ref structural sequence, but its frame can be shifted without affecting Ref activity. The leader region ends with an apparent rho-independent termination sequence (attenuator). Deletion of 18 bp of early leader sequence drastically reduced Ref activity, even when ref was driven by a heterologous promoter (plac). An 8-bp internal deletion in the putative attenuator sequence relieved this requirement for the early leader sequence. This latter observation, along with nucleotide complementarity between portions of the early leader and attenuator sequences, are consistent with preemption of attenuation by the early leader.