Identification of the RNA Binding Domain of T4 RegA Protein by Structure-based Mutagenesis

• Published in: The Journal of biological chemistry 1999-11, Vol.274 (45), p.32265-32273
• Main Authors: Gordon, Johnthan, Sengupta, Tapas K., Phillips, Christine A., O'Malley, Shawn M., Williams, Kenneth R., Spicer, Eleanor K.
• Format: Article
• Language: English
• Subjects: Bacteriophage T4 - genetics; Bacteriophage T4 - metabolism; Binding Sites; Circular Dichroism; Models, Molecular; Mutagenesis, Site-Directed; Phage T4; Protein Conformation; RegA protein; RNA - metabolism; RNA-binding protein; Structure-Activity Relationship; Viral Proteins - genetics; Viral Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.45.32265

The T4 translational repressor RegA protein folds into two structural domains, 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 β-sheet and two α-helices. Domain II contains a four-stranded β-sheet and an unusual 3/10 helix. Since β-sheet residues play a role in a number of protein-RNA interactions, one or both of the β-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both β-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of β-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an α-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.