THE ACTION OF POKEWEED ANTIVIRAL PROTEIN AND RICIN A-CHAIN ON MUTANTS IN THE ALPHA-SARCIN LOOP OF ESCHERICHIA-COLI 23S RIBOSOMAL-RNA

The alpha-sarcin loop of Escherichia coli 23S rRNA is a universally-conserved structure involved in the binding of elongation factors Tu and G and is the site of action of the ribosome-inactivating proteins (RIPs). One such group, the N-glycosidase RIPs, act by the removal of a single adenine residue (A2660) believed to exist in a GAGA-containing tetraloop structure. The action of two RIPs, the catalytic A-chain from the heterodimeric toxic lectin ricin (RTA) and the single-chain RIP pokeweed antiviral protein (PAP), which are known to be highly homologous in tertiary structure, was determined on native ribosomes or naked 23S rRNA containing mutations designed to affect the structure of the GAGA tetraloop. One such mutant rRNA containing G2663C, which abolishes the potential tetraloop by disrupting the Watson-Crick base-pair involved in closing it; resulted in a loss of depurination by RTA, but not by PAP. A similar result was observed for mutant G2661A. The double mutant C2658G + G2663C, which restores the tetraloop-closing base-pair but in the reverse orientation, resulted in sensitivity to both PAP and RTA, as in the wild-type. Thus, the tetraloop structure is required for the action of RTA, but not of PAP, and unlike RTA, PAP does not require G at position 2661. RNA containing the G2664C mutation, which lies outside the tetraloop, served as a substrate for both PAP and RTA. The comparison of the recognition elements for PAP and RTA was made with naked (deproteinised) rRNA, because RTA does not act on E, coil ribosomes. However, PAP is active on E. coli ribosomes, and it was found that the action of PAP on ribosomes containing the above mutations paralleled exactly that on the corresponding naked rRNAs. It is concluded that the recognition elements for PAP and RTA differ and may account, at least in part, for the fact that PAP but not RTA catalyses the depurination of E. coli ribosomes. (C) 1995 Academic Press Limite