BACTERIOPHAGE-T4 REGA PROTEIN BINDS RNA AS A MONOMER, OVERCOMING DIMER INTERACTIONS

The stoichiometry of the complex formed between the T4 translational r epressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA, It is known that regA pro tein exists as a dimer in protein crystals, Thus, the stoichiometry ma y be indicative of a regA dimer bound to two RNAs or a regA monomer bo und to one RNA, Gel filtration through Sephadex G-75 revealed that wil d-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, co nsistent with the mass of a dimer. However, wildtype regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at similar to 20 kDa, consistent with a regA monomer-RNA complex, Covalent crosslink ing of surface lysines with glutaraldehyde confirmed that wild-type an d R91L proteins exist as dimers and higher oligomers in solution, Howe ver, the addition of RNA to wild-type regA protein prior to crosslinki ng inhibited the formation of crosslinked dimers, Thus, the regA prote in-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA, Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.