Determination of the 28 S Ribosomal RNA Identity Element (G4319) for Alpha-sarcin and the Relationship of Recognition to the Selection of the Catalytic Site

Abstract

Ricin A-chain and α-sarcin are ribotoxins that inactivate eukaryotic ribosomes by modifying 28 S rRNA; ricin A-chain is an RNA N-glycosidase that depurinates the adenosine at position 4324 and α-sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3 ′ side of the adjacent guanosine (at position 4325). In cartoons of the secondary structure these two residues are seen to be embedded in a 17 base single-stranded loop over a seven base-pair helix. However, NMR spectroscopy of an oligoribonucleotide, a 29-mer that mimics the sarcin/ricin domain, indicates that the RNA has a compact conformation in which the guanosine at the position analogous to 4319 in 28 S rRNA is bulged out of what otherwise is an extended A-form helix. Since similar structural irregularities are used by proteins to bind to RNA, we have tested the effect of mutations of the bulged guanosine on recognition and covalent modification of the RNA by ricin A-chain and by α-sarcin. For the test a synthetic oligoribonucleotide, a 35-mer, was used; the mutations were the deletion, the transition to adenosine, and the transversions to cytidine and uridine of the guanosine that is the analog of G4319. Each of the four mutations abolished cleavage of the RNA by α-sarcin, whereas depurination by ricin A-chain was little affected. Thus G4319 is an identity element for α-sarcin recognition. Analysis of the effect of α-sarcin on variant oligoribonucleotides in which additional bases were inserted between the identity element guanosine and the site of catalysis suggest that on binding to the RNA the toxin uses the guanosine for orientation and then cleaves at a fixed distance and at a fixed position in space.