Regions of bacteriophage T4 and RB69 RegA translational repressor proteins that determine RNA-binding specificity

RegA protein of T4 and related bacteriophages is a highly conserved RNA-binding protein that represses the translation of many phage mRNAs that encode enzymes involved in DNA metabolism. RB69, a T4-related bacteriophage, has a unique regA gene, which we have cloned, sequenced, and expressed. The predicted amino acid sequence of RB69 RegA is 78% identical to that of T4 RegA. Plasmid-encoded RB69 RegA expressed in vivo represses the translation of T4 early mRNAs, including those of rIIA, rIIB, 44, 45, rpbA, and regA. Nucleotide sequences were determined for several T4 and RB69 regA mutations, and their corresponding repressor properties were characterized. All of the 10 missense mutations affect residues conserved between RB69 and T4 RegA. Two regions of RegA are especially sensitive to mutation: one between Val-15 and Ala-25 and another between Arg-70 and Ser-73. Sequence alignments and mutational data suggest that the region from Val-15 to Ala-25 is similar to helix-turn-helix domains of DNA-binding proteins and confers RNA-binding specificity upon RegA. The RegA691 protein (Ile-24----Thr) has an in vivo phenotype that appears to distinguish site-specific and cooperative binding modes of hierarchical RegA-mediated translational repression.