Mutational analysis of the protein subunits of the signal recognition particle Alu-domain.
Two polypeptides of the murine signal recognition particle (SRP), SRP9 and SRP14, bind exclusively as a heterodimer to SRP RNA and their presence is required for elongation arrest activity of the particle. SRP9/14 also constitute a subunit of small cytoplasmic Alu RNPs. To identify RNA-binding determinants, we assayed the dimerization and RNA-binding capacities of altered proteins in vitro. Despite the structural homology of the two proteins, their requirements for dimerization differ substantially. In SRP9, an internal fragment of 43 amino acids is sufficient to allow dimer formation, whereas in SRP14 only few changes, such as removing an internal loop region, are tolerated without affecting its dimerization activity. The dimerization defect of the SRP14 proteins is most likely explained by a reduced stability or ability to fold of the proteins. Interestingly, SRP RNA can engage certain dimerization-defective SRP14 proteins into stable complexes, suggesting that low-affinity interactions between the RNA and SRP14 may help to overcome the folding defect or the reduced stability of the proteins. We identified two regions, one in each protein, that are essential for RNA-binding. In SRP9, acidic amino acid residues in the N-terminal alpha-helix 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 being formed by both proteins.
Bui
N
N
Departement de biologie cellulaire, Université de Genève, Sciences III, Geneva, Switzerland.
Wolff
N
N
Cusack
S
S
Strub
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004252:DNA Mutational Analysis; D019281:Dimerization; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D018271:Signal Recognition Particle / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018271:Signal Recognition Particle; C490325:Srp14 protein, mouse; C503637:Srp72 protein, mouse; D012313:RNA