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 d uring translation leads to an inhibition of polypeptide elongation kno wn as translation arrest, The arrest activity is mediated by a discret e domain comprised of the Alu portion of SRP RNA and a 9 and 14 kDa po lypeptide 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 tha t 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 b ind 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 similar to 2- and 5-fold respectively, The mutan t RNAs could nevertheless assemble into SRPs at high subunit concentra tions, SRPs reconstituted with mutant RNAs were not significantly defe ctive in translation arrest assays, indicating that the conserved guan osine does not interact directly with the translational machinery, Tak en together, these results demonstrate that G24 plays an important rol e in the translation arrest function of SRP by mediating high affinity binding of SRP9/14.