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Role of an active site adenine in hairpin ribozyme catalysis

Academic Article
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Overview

authors

  • Kuzmin, Y. I.
  • Da Costa, C. P.
  • Cottrell, J. W.
  • Fedor, Martha

publication date

  • June 2005

journal

  • Journal of Molecular Biology  Journal

abstract

  • The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.

subject areas

  • Adenine
  • Base Pairing
  • Base Sequence
  • Binding Sites
  • Catalysis
  • Guanine
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Conformation
  • Oligonucleotides
  • RNA, Catalytic
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Research

keywords

  • RNA catalysis
  • abasic ribozyme
  • catalytic mechanism
  • exogenous nucleobase rescue
  • hairpin ribozyme
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Identity

International Standard Serial Number (ISSN)

  • 0022-2836

Digital Object Identifier (DOI)

  • 10.1016/j.jmb.2005.04.005

PubMed ID

  • 15907933
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Additional Document Info

start page

  • 989

end page

  • 1010

volume

  • 349

issue

  • 5

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