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Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme.

Publication ,  Journal Article
Perrotta, AT; Wadkins, TS; Been, MD
Published in: RNA
July 2006

In the ribozyme from the hepatitis delta virus (HDV) genomic strand RNA, a cytosine side chain is proposed to facilitate proton transfer in the transition state of the reaction and, thus, act as a general acid-base catalyst. Mutation of this active-site cytosine (C75) reduced RNA cleavage rates by as much as one million-fold, but addition of exogenous cytosine and certain nucleobase or imidazole analogs can partially rescue activity in these mutants. However, pH-rate profiles for the rescued reactions were bell shaped, and only one leg of the pH-rate curve could be attributed to ionization of the exogenous nucleobase or buffer. When a second potential ionizable nucleobase (C41) was removed, one leg of the bell-shaped curve was eliminated in the chemical-rescue reaction. With this construct, the apparent pK(a) determined from the pH-rate profile correlated with the solution pK(a) of the buffer, and the contribution of the buffer to the rate enhancement could be directly evaluated in a free-energy or Brønsted plot. The free-energy relationship between the acid dissociation constant of the buffer and the rate constant for cleavage (Brønsted value, beta, = approximately 0.5) was consistent with a mechanism in which the buffer acted as a general acid-base catalyst. These data support the hypothesis that cytosine 75, in the intact ribozyme, acts as a general acid-base catalyst.

Duke Scholars

Published In

RNA

DOI

ISSN

1355-8382

Publication Date

July 2006

Volume

12

Issue

7

Start / End Page

1282 / 1291

Location

United States

Related Subject Headings

  • RNA, Viral
  • RNA, Catalytic
  • Nucleic Acid Conformation
  • Mutation
  • Molecular Sequence Data
  • Kinetics
  • Hydrogen-Ion Concentration
  • Hepatitis Delta Virus
  • Genome, Viral
  • Developmental Biology
 

Citation

APA
Chicago
ICMJE
MLA
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Perrotta, A. T., Wadkins, T. S., & Been, M. D. (2006). Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme. RNA, 12(7), 1282–1291. https://doi.org/10.1261/rna.14106
Perrotta, Anne T., Timothy S. Wadkins, and Michael D. Been. “Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme.RNA 12, no. 7 (July 2006): 1282–91. https://doi.org/10.1261/rna.14106.
Perrotta, Anne T., et al. “Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme.RNA, vol. 12, no. 7, July 2006, pp. 1282–91. Pubmed, doi:10.1261/rna.14106.

Published In

RNA

DOI

ISSN

1355-8382

Publication Date

July 2006

Volume

12

Issue

7

Start / End Page

1282 / 1291

Location

United States

Related Subject Headings

  • RNA, Viral
  • RNA, Catalytic
  • Nucleic Acid Conformation
  • Mutation
  • Molecular Sequence Data
  • Kinetics
  • Hydrogen-Ion Concentration
  • Hepatitis Delta Virus
  • Genome, Viral
  • Developmental Biology