Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif

Double-stranded RNA-binding proteins constitute a large family with conserv ed domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TA R RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequenc e homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishe s RNA binding, suggesting that only one domain is functional. To analyse fu rther the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10(-8) m, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and othe r related proteins showed that only one peptide in TRBP and one in Drosophi la Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide d erived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (T R6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Anal ysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRB P dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basi s of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required f or binding TAR. This structural motif exists independently from the dsRBD c ontext and therefore has a modular function.