MUTAGENESIS OF THE COOH-TERMINAL REGION OF BACTERIOPHAGE-T4 REGA PROTEIN

The bacteriophage T4 regA protein is a translational repressor that re gulates the synthesis of >12 T4 proteins. Earlier studies demonstrated that photo-cross-linking of the 122-residue regA protein to (dT)(16) occurs at two sites, with the major site occurring at Phe-106. Amino a cid substitutions were introduced at Phe-106 to evaluate its role in n ucleic acid binding. Binding affinities of mutants F106C, F106V, and F 106Y for nonspecific and specific RNA ligands indicated little differe nce between the K-app of the mutants and wild type regA protein, for e ither poly(U) or for a specific gene 44 oligoribonucleotide. Thus, Phe -106 does not contribute measurably to the overall free energy of bind ing. Partial proteolysis of regA protein was carried out to further pr obe its domain structure, Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein. Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RN A. Two deletion mutants, 1-->94 and 1-->109, have also been cloned and purified. The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U). These studi es indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding.