A. Lambdoid phage HK002, like its relatives, contains transcription terminators in its two early operons and expresses genes located downstream of these terminators by antiterminating transcription. The mechanism of HKO22 antitermination appears simpler than that of its relatives because HKO22 requires no phage-encoded protein and appears to require no host-encoded proteins besides RNA polymerase itself. We have isolated host mutants that are unable to antiterminate HKO22 early transcription. All of 14 independently isolated mutations change amino acids near the amino terminus of the beta subunit of host RNA polymerase. In vitro transcription studies with purified enzyme revealed that wild type polymerase read through a strong transcription terminator with high efficiency (about 50%) in a template containing a PL-proximal HKO22 sequence that is required for antitermination in vivo. Read- through was much less efficient when we used polymerase with a mutant beta subunit or a template without the promoter-proximal sequence. We speculate that interaction of the amino-terminal region of the RNA polymerase beta subunit with sequences lying within or near the HKO22 early promoters effects antitermination. B. The integrase proteins of phages lambda and HKO22 are site specific recombinases that are closely related in structure and function, but recognize different nucleotide sequences in the crossover regions of the attachment sites of the two phages. It is not known what region of Int recognizes the crossover region or how this region is related to the catalytic site. In order to locate the parts of the proteins that discriminate between the substrates, we selected mutations that allowed lambda Int to recombine HKO22 attachment sites. Most of the altered proteins had relaxed specificity; i.e., they retained the ability to recombine the original attachment sites. One mutation altered specificity; i.e., it no longer recombined the original sites. This mutation changes Asn99 of lambda Int to Asp, the corresponding HKO22 residue. We have developed new recombination substrates that will facilitate isolation of such specificity mutants, and should allow us to determine the identity of the amino acid-nucleotide interaction.
