| Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue. | |
MedLine Citation:
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PMID: 15201049 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state. |
Authors:
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Yaroslav I Kuzmin; Carla P Da Costa; Martha J Fedor |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Journal of molecular biology Volume: 340 ISSN: 0022-2836 ISO Abbreviation: J. Mol. Biol. Publication Date: 2004 Jul |
Date Detail:
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Created Date: 2004-06-17 Completed Date: 2004-07-21 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 2985088R Medline TA: J Mol Biol Country: England |
Other Details:
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Languages: eng Pagination: 233-51 Citation Subset: IM |
Affiliation:
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Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB35, La Jolla, CA 92037, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Base Sequence Binding Sites Catalysis Guanine / metabolism* Hydrogen-Ion Concentration Hydrolysis Molecular Probes Molecular Sequence Data Nucleic Acid Conformation RNA, Catalytic / chemistry, metabolism* |
| Grant Support | |
ID/Acronym/Agency:
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R01 GM46422/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Molecular Probes; 0/RNA, Catalytic; 73-40-5/Guanine |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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