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 ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis 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 with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.
Skinner
L M
LM
Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Jackson
M P
MP
D000225:Adenine / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D002522:Chlorocebus aethiops; D018274:Electroporation; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism*; D022603:Shiga Toxins; D012760:Shigella; D014709:Vero Cells
D001427:Bacterial Toxins; D012339:RNA, Ribosomal, 28S; D022603:Shiga Toxins; D012343:RNA, Transfer; D000225:Adenine