Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization

Garrett Soukup, Ronald R. Breaker

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

Background: Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. Results: In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G·U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the Wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of ~40-fold in the presence of theophylline. Conclusions: An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.

Original languageEnglish
Pages (from-to)783-791
Number of pages9
JournalStructure
Volume7
Issue number7
DOIs
StatePublished - Jul 15 1999

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Catalytic RNA
Ligands
Flavin Mononucleotide
Theophylline
Catalytic Domain
RNA
Allosteric Site
hammerhead ribozyme
Enzymes
Nucleic Acids

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Structural Biology

Cite this

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization. / Soukup, Garrett; Breaker, Ronald R.

In: Structure, Vol. 7, No. 7, 15.07.1999, p. 783-791.

Research output: Contribution to journalArticle

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