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| The EMBO Journal
(2001) 20, 4884–4891, doi:10.1093/emboj/20.17.4884 |
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| Energetic contribution of non-essential 5' sequence to catalysis in a hepatitis delta virus ribozyme |
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| I.-hung Shih2 and Michael D. Been1 |
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1 Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA 2 Present address: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA To whom correspondence should be addressed Michael D. Been, been@biochem.duke.edu Received 22 February 2001; Revised 5 July 2001; Accepted 12 July 2001. |
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| Abstract |
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| Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid–base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5' sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5' sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb's free energy of substrate binding revealed a linear relationship with a slope of -1. This relationship is consistent with a model in which components of the substrate immediately 5' to the cleavage site in the HDV ribozyme–substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme. |
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| Keywords: catalytic mechanism, catalytic RNA, ground-state destabilization, HDV ribozyme, self-cleaving RNA |
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