MUTATIONAL ANALYSIS OF THE PROTEIN SUBUNITS OF THE SIGNAL RECOGNITIONPARTICLE ALU-DOMAIN

Two polypeptides of the murine signal recognition particle (SRP), SRP9 and SRP14, bind exclusively as a heterodimer to SRP RNA and their pre sence is required for elongation arrest activity of the particle, SRP9 /14 also constitute a subunit of small cytoplasmic Alu RNPs. To identi fy RNA-binding determinants, we assayed the dimerization and RNA-bindi ng capacities of altered proteins in vitro. Despite the structural hom ology of the two proteins, their requirements for dimerization differ substantially. In SRP9, an internal fragment of 43 amino acids is suff icient to allow dimer formation, whereas in SRP14 only few changes, su ch as removing an internal loop region, are tolerated without affectin g its dimerization activity. The dimerization defect of the SRP14 prot eins is most likely explained by a reduced stability or ability to fol d of the proteins. Interestingly, SRP RNA can engage certain dimerizat ion-defective SRP14 proteins into stable complexes, suggesting that lo w-affinity interactions between the RNA and SRP14 may help to overcome the folding defect or the reduced stability of the proteins, We ident ified two regions, one in each protein, that are essential for RNA-bin ding. in SRP9, acidic amino acid residues in the N-terminal alpha-heli x and the adjacent loop and, in SRP14, a flexible internal loop region are critical for RNA-binding. In the heterodimer, the two regions are located in close proximity, consistent with the RNA-binding region be ing formed by both proteins.