Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA.
The interaction of protein SRP54M from the human signal recognition particle with SRP RNA was studied by systematic site-directed mutagenesis of the RNA molecule. Protein binding sites were identified by the analysis of mutations that removed individual SRP RNA helices or disrupted helical sections in the large SRP domain. The strongest effects on the binding activity of a purified polypeptide that corresponds to the methionine-rich domain of SRP54 (SRP54M) were caused by changes in helix 8 of the SRP RNA. Binding of protein SRP19 was diminished significantly by mutations in helix 6 and was stringently required for SRP54M to associate. Unexpectedly, mutant RNA molecules that resembled bacterial SRP RNAs were incapable of interaction with SRP54M, showing that protein SRP19 has an essential and direct role in the formation of the ternary complex with SRP54 and SRP RNA. Our findings provide an example for how, in eukaryotes, an RNA function has become protein dependent.
Gowda
K
K
Department of Molecular Biology, The University of Texas Health Science Center at Tyler, PO Box 2003, Tyler, TX 75710, USA.
Chittenden
K
K
Zwieb
C
C
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism
D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D018271:Signal Recognition Particle; D012313:RNA