Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena.

Abstract:

:Like protein enzymes, RNA enzymes (ribozymes) provide specific binding sites for their substrates. We now show that equilibrium dissociation constants for complexes between the Tetrahymena ribozyme and its RNA substrates and products can be directly measured by electrophoresis in polyacrylamide gels containing divalent cations. Binding is 10(3)- to 10(4)-fold tighter (4-5 kcal/mol at 42 degrees C) than expected from base-pairing interactions alone, implying that tertiary interactions also contribute to energetic stabilization. Binding decreases with single base changes in the substrate, substitution of deoxyribose sugars, and lower Mg2+ concentration. Ca2+, which enables the ribozyme to fold but is unable to mediate efficient RNA cleavage, promotes weaker substrate binding than Mg2+. This indicates that Mg2+ has special roles in both substrate binding and catalysis. Mutagenesis of a region near the internal guide sequence disrupts substrate binding, whereas binding is not significantly affected by a mutation of the guanosine-binding site. This approach should be generally useful for analysis of ribozyme variants independent of their catalytic activities.

authors

Pyle AM,McSwiggen JA,Cech TR

doi

10.1073/pnas.87.21.8187

subject

Has Abstract

pub_date

1990-11-01 00:00:00

pages

8187-91

issue

21

eissn

0027-8424

issn

1091-6490

journal_volume

87

pub_type

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