INVESTIGATION OF RIBOSOME BINDING BY THE SHIGA TOXIN A1 SUBUNIT, USING COMPETITION AND SITE-DIRECTED MUTAGENESIS

The enzymatic subunit of Shiga toxin (StxA1) is a member of the riboso me-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depuri nates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the a cceptor site of eukaryotic ribosomes, The specific activities of nativ e StxA1, as well as mutated forms of the enzyme with substitutions in catalytic Bite residues, were measured by an in vitro translation assa y, Electroporation was developed as an alternative method for the deli very of purified A1 polypeptides into Vero cells. Site-directed mutage nesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering w ith the capacity of StxA1 to depurinate 28S rRNA, Ribosomes were prote cted from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior t o binding and depurinating the GAGA tetra-loop.