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RNA. 2014 Nov;20(11):1732-46. doi: 10.1261/rna.044362.114. Epub 2014 Sep 22.

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction.

Author information

1
Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA.
2
Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA herschla@stanford.edu.

Abstract

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5'-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5'-exon intermediate in self splicing to remain bound subsequent to 5'-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.

KEYWORDS:

Group I intron; catalytic framework; noncoding RNA; ribozyme

PMID:
25246656
PMCID:
PMC4201826
DOI:
10.1261/rna.044362.114
[Indexed for MEDLINE]
Free PMC Article

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