Anti-termination increases the transcription of genes that are located downstream of terminators. Transcripts encoded by the cis-acting antitermination sites (put) of lambdoid phage HK022 promote readthrough of multiple downstream transcription terminators. Efficient readthrough depends on the structure of the put transcript and of a domain located in the largest subunit of RNA polymerase, suggesting that an interaction between put RNA and this domain is a step in anti-termination. To study put structure and function, we characterized a large number of mutants and probed synthetic transcripts with structure-specific nucleases. The results argue that the secondary structure of active put RNA consists of two hairpin stems that are separated by a single unpaired base. Internal loops and bulges in one of the stems are important for activity. Some variation in the lengths of the stems, the sizes and sequences of the terminal loops, and the locations of the internal loops and bulges is allowed. We propose that put acts by preventing dissociation of the ter nary transcription complex at terminator sites, because a terminator that arrests polymerase but does not cause dissociation is resistant to put. The integrase proteins of phages lambda and HK022 are closely related site- specific recombinases that recognize different nucleotide sequences in the core regions of their substrates, the attachment sites of the two phages. In lambda attachment sites, -2C, -1T, and +4G of the B' extended core binding site and -1T of C are major anti-HK022 determinants. In HK022 attachment sites, +1A and +3C of B' and C are major anti-? determinants. These determinants are best viewed as negative because when they were replaced with the corresponding bases from the other attachment site, specificity was relaxed. Residues at certain positions within the two integrases were identified as specificity determinants. We have now shown that the phenotypes of amino acid substitutions at these positions can be suppressed in an allele-specific manner by nucleotide substitutions in the attachment sites, suggesting a direct and specific amino acid-nucleotide interaction.
