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0344760467
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unpublished data
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a of the base and that, in the precursor, the N-3 of γC75 may be positioned to accept the proton from the 2′-hydroxyl group of the ribose at position -1.
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a of the base and that, in the precursor, the N-3 of γC75 may be positioned to accept the proton from the 2′-hydroxyl group of the ribose at position -1.
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32P]triphosphate and polynucleotide kinase after dephosphorylating with calf intestinal phosphatase (6). The mutations at position 76 and modifications to the sequence 5′ to the cleavage site were generated by oligonucleotide- directed mutagenesis on single-stranded uracil-containing templates of the plasmid (6).
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0344328419
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note
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-1). Buffers that were tested and failed to support activity were tris-HCl, Hepes, Pipes, and MES at pH 7.5; CHES at pH 8.5; and sodium acetate at a variety of pH values. Other potential bases - ethylenediamine (pH 7.8), pyridine (pH 7.5), and ammonia (ammonium acetate, pH 8.5) - failed to rescue cleavage activity. The tris buffer did not inhibit imidazole-dependent cleavage of the C76u ribozyme. However, significant stimulation by imidazole was not a general phenomenon for the HDV antigenomic ribozyme. With the wild-type ribozyme, a moderate increase in the rate constant (1.2-fold) occurred with the addition of 200 mM imidazole (pH 7.4) to the reaction mixture (Table 1). We observed no stimulation of cleavage activity with the C76a ribozyme.
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19
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0029859191
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31
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0344760443
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note
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Supported by NIH grant GM47322. We thank T. Cech, S. Crary, C. Fierke, D. Herschlag, T.-s. Hsieh, and T. Wadkins for critical comments and helpful suggestions.
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