ANALYSIS OF THE CLEAVAGE REACTION OF A TRANS-ACTING HUMAN HEPATITIS-DELTA VIRUS RIBOZYME

The cleavage reaction catalyzed by the transacting genomic ribozyme of human hepatitis delta virus (HDV) was analyzed with a 13mer substrate (R13) and thio-substituted [SR13(Rp) and SR13(Sp)] substrates under s ingle-turnover conditions. The cleavage of RNA by the transacting HDV ribozyme proceeded as a first order reaction, The logarithm of the rat e of cleavage (k(clv)) increased linearly (with a slope of similar to 1) between pH 4.0 and 6.0, an indication that a single deprotonation r eaction occurred, This result suggests that k(clv) reflects the rate o f the chemical cleavage step, at least around pH 5, The amount of acti ve complex with the SR13(Sp) substrate was almost as large as with R13 (60-80%), whereas the amount of the corresponding active complex form ed with the SR13(Rp) substrate was, at most, 20% of this value (with 0 .5-100 mM Mg2+ ions) at pH 5.0, Nonetheless, the value of k(clv) for a ll substrates was almost the same (0.4-0.5 min(-1)), Neither a 'thio e ffect' nor a 'Mn2+ rescue effect' were observed, These results suggest that Mg2+ ions do not interact with pro-R oxygen directly but are ess ential to the formation of the active complex of the ribozyme and its substrate.