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 conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further 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 other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) 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. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis 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 for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.
Daviet
L
L
U332 INSERM, U529 INSERM, Institut Cochin de Génétique Moléculaire, Paris, France.
Erard
M
M
Dorin
D
D
Duarte
M
M
Vaquero
C
C
Gatignol
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D017124:Conserved Sequence; D004330:Drosophila / Q000737:chemistry; D015497:HIV-1 / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D019893:Ribosomal Protein S6 Kinases / Q000737:chemistry; D000099253:Trans-Activation Responsive RNA-Binding Protein
D010446:Peptide Fragments; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D019893:Ribosomal Protein S6 Kinases; D000099253:Trans-Activation Responsive RNA-Binding Protein