Mechanism of Action of Bacteriophage T4 Translational Repressor regA Protein.

摘要

The bacteriophage T4 regA protein (M sub r=14,600) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to (32P)p(dT)16. Two tryptic peptides cross-linked to (32P)p(dT)16 were isolated, and sequencing of the major cross-linked peptide yielded the sequence VISXKQKHEWK, corresponding to residues 103-113. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein:p(dT)16 complex. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to (32P)p(dT)16 and 70% of the non-specific binding energy of the intact protein. Site directed mutagenesis has been used to introduce substitutions at Phe 106. Studies to measure the contribution of Phe 106 to the free energy of RNA binding are in progress.