A ribozyme model: site-specific cleavage of an RNA substrate by Mn2+.
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Biomedical subjects
R B Van Atta
Publications and source records attributed to R B Van Atta.
On the chemical nature of DNA and RNA modification by a hemin model system.
In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)
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A Mn2(+)-dependent ribozyme.
An RNA hairpin identical in sequence with the one formed during autocyclization of the 414-nucleotide Tetrahymena intervening sequence undergoes strand scission at a specific site in the presence of Mn2+. In addition to representing one of the smallest and simplest ribozymes possible, strand scission occurs readily under physiological conditions, is unaffected by the presence of Mg2+, and displays salt, pH, and temperature optima of potential use in exploiting Mn2+ as a regulatory switch in intact cells. The chemistry of strand scission of the RNA hairpin is described, as is the Mn2(+)-dependent solvolysis of a 231-nucleotide RNA transcript containing this structural motif.
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