A highly conserved nucleotide in the Alu domain of SRP RNA mediates translation arrest through high affinity binding to SRP9/14.
Binding of the signal recognition particle (SRP) to signal sequences during translation leads to an inhibition of polypeptide elongation known as translation arrest. The arrest activity is mediated by a discrete domain comprised of the Alu portion of SRP RNA and a 9 and 14 kDa polypeptide heterodimer (SRP9/14). Although very few nucleotides in SRP RNA are conserved throughout evolution, the remarkable conservation of G24, which resides in the region of SRP9/14 interaction, suggests that it is essential for translation arrest. To understand the functional significance of the G24 residue, we made single base substitutions in SRP RNA at this position and analyzed the ability of the mutants to bind SRP9/14 and to reconstitute functional SRPs. Mutation of G24 to C reduced binding to SRP9/14 by at least 50-fold, whereas mutation to A and U reduced binding approximately 2- and 5-fold respectively. The mutant RNAs could nevertheless assemble into SRPs at high subunit concentrations. SRPs reconstituted with mutant RNAs were not significantly defective in translation arrest assays, indicating that the conserved guanosine does not interact directly with the translational machinery. Taken together, these results demonstrate that G24 plays an important role in the translation arrest function of SRP by mediating high affinity binding of SRP9/14.
Chang
D Y
DY
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Newitt
J A
JA
Hsu
K
K
Bernstein
H D
HD
Maraia
R J
RJ
D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D017931:DNA Primers; D019281:Dimerization; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D018271:Signal Recognition Particle