Site-directed mutagenesis of signal-recognition particle RNA. Identification of the nucleotides in helix 8 required for interaction with protein SRP19.

Abstract

The RNA component of signal recognition particle (SRP) consists of eight helices which form a functional unit with the proteins of the SRP. The primary binding site of the 19-kDa protein of SRP (SRP19) is a tetranucleotide loop (tetraloop) in helix 6 of the SRP RNA, but additional determinants are located in helix 8, which might play important roles in the assembly and the function of the particle. To determine the structural features in helix 8 essential for interaction with SRP19, we altered helix 8 systematically by site-directed mutagenesis, and determined the ability of protein SRP19 to interact with the various mutant SRP RNAs. Binding of SRP19 was affected by base changes introduced into the 5' portion (192A, 193G, 194G in the human SRP RNA), but not into the 3' portion (205 A, 206G, 207C) of the distally located conserved internal loop of helix 8. Of the three bases at positions 192-194, only a pyrimidine at position 192 impaired the association with SPR19. An important feature of the SRP19-RNA interaction were the three base pairs U195-G204, C196-G203 and G197-C202 which shape the helix-8 tetraloop. Some base-specific features in the base pairs were also recognized. The tetraloop bases of helix 8 were dispensable for the interaction with SRP19.