Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme.
The cleavage reaction catalyzed by the trans -acting 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 single-turnover conditions. The cleavage of RNA by the trans -acting HDV ribozyme proceeded as a first order reaction. The logarithm of the rate of cleavage (kclv) increased linearly (with a slope of approximately 1) between pH 4.0 and 6.0, an indication that a single deprotonation reaction occurred. This result suggests that kclv reflects the rate of the chemical cleavage step, at least around pH 5. The amount of active complex with the SR13(Sp) substrate was almost as large as with R13 (60-80%), whereas the amount of the corresponding active complex formed 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 kclv for all substrates was almost the same (0.4-0.5 min-1). Neither a 'thio effect' nor a 'Mn2+rescue effect' were observed. These results suggest that Mg2+ions do not interact with pro-R oxygen directly but are essential to the formation of the active complex of the ribozyme and its substrate.
Fauzi
H
H
National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Tsukuba Science City 305, Japan.
Kawakami
J
J
Nishikawa
F
F
Nishikawa
S
S
D001483:Base Sequence; D002384:Catalysis; D002413:Cations, Divalent; D003698:Hepatitis Delta Virus / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D002413:Cations, Divalent; D016337:RNA, Catalytic; D015534:Trans-Activators; D008345:Manganese; D008274:Magnesium