Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P

Abstract

The ribozyme from bacterial ribonuclease P recognizes two structural modules in a tRNA substrate: the T stem-loop and the acceptor stem. These two modules are connected through a helical linker. The T stem-loop binds at a surface confined in a folding domain away from the active site. Substrates for the Bacillus subtilis RNase P RNA were previously selected in vitro that are shown to bind comparably well or better than a tRNA substrate. Chemical modification of P RNA-substrate complexes with dimethylsulfate and kethoxal was performed to determine how the P RNA recognizes three in vitro selected substrates. All three substrates bind at the surface known to interact with the T stem-loop of tRNA. Similar to a tRNA, the secondary structure of these substrates contains a helix around the cleavage site and a hairpin loop at the corresponding position of the T stem-loop. Unlike a tRNA, these two structural modules are connected through a non-helical linker. The two structural modules in the tRNA and in the selected substrates bind to two different domains in P RNA. The properties of substrate recognition exhibited by this ribozyme may be exploited to isolate new ribozyme-substrate pairs with interactive structural modules.