Modular construction of a tertiary RNA structure: The specificity domain of the Bacillus subtilis RNase P RNA

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress modu le, analogous to the bipartite structure of the P4-P6 domain of the Tetrahy mena group I ribozyme. The core module is the functional unit of the S-doma in and contains the binding site for the T stem-loop of a tRNA. The buttres s module provides structural stability to the core module and consists of a GA(3) tetraloop and its receptor. To explicitly test the hypothesis that m odular construction can describe the structure of the S-domain and is a use ful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a bas e pair in the helical linker region between the modules only modestly desta bilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetralo op-receptor interactions significantly destabilizes the core module and res ults in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivi ty. Overall, we have observed that the linker regions between the two modul es can tolerate moderate structural changes and that the buttress modules c an be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functio nal module.