Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.
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 beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein 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 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 beta-sheet residues. Instead, the RNA binding 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 properties.
Gordon
J
J
Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Sengupta
T K
TK
Phillips
C A
CA
O'Malley
S M
SM
Williams
K R
KR
Spicer
E K
EK
D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C066700:RegA protein, Enterobacteria phage T4; D014764:Viral Proteins; D012313:RNA