In vitro selection of phage RB69 RegA RNA binding sites yields UAA triplets

The SELEX method of in vitro selection was used to isolate RNAs that bind the RB69 RegA translational repressor protein immobilized on Ni-NTA agarose. After five rounds of SELEX, the pool of selected RNA displayed striking sequence uniformity: UAAUAAUAAUAAUA was clearly enriched in the 14 nucleotides that underwent selection. Individual, cloned molecules displayed a repeating (UAA) sequence, with only two RNAs having a 3' AUG. Removing the 3' AUG slightly reduced binding in gel shift assays, moving the AUG 5' proximal of the (UAA) slightly improved binding, but (UAA)4 alone still bound the purified protein. Dissociation constants showed that RNA shortened to (UAA)3 and (UAA)2 also retained binding, whereas cytosine clearly prevented binding by RB69 RegA. Scanning of RB69 gene starts and ends with an RB69 RegA SELEX information weight matrix yielded 21 sequences as potential RegA sites. One site, on the mRNA for the pentameric (4:1) phage gp44/62 DNA polymerase clamp loader complex, has the RB69 gene 44 stop codon and 3'-adjacent gene 62 initiation codon in a sequence (GAAAUAAUAUG) that is similar to in vitro selected RNA and was shown to bind RB69 RegA. Sequences between the Shine-Dalgarno and initiation codon, which frequently contain a UAA stop codon of a 5'-adjacent gene, appear to be preferred RB69 RegA binding sites.