Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis

The T4 translational repressor RegA protein folds into two structural domai ns, as revealed by the crystal structure (Kang, C.-H., Chan, R., Berger, I. , Lockshin, C., Green, L,, Gold, L,, and Rich, A. (1995) Science 268, 1170- 1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an un usual 3/10 helix, Since beta-sheet residues play a role in a number of prot ein-RNA interactions, one or both of the beta-sheet regions in Reg-A protei n may be involved in RNA binding, To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA bind ing affinities of RegA protein were measured. Additional sites for mutagene sis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluores cence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shi ft assays using lysed cell supernatants, The results of this mutagenesis st udy ruled out the participation of beta-sheet residues. Instead, the RNA bi nding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition p roperties.