Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes

Timothy J. Wilson, Yijin Liu, Nan-Sheng Li, Qing Dai, Joseph A. Piccirilli, David M. J. Lilley (Lead / Corresponding author)

Research output: Contribution to journalArticle

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Abstract

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2′ nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2′-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2′ of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct.

Original languageEnglish
Pages (from-to)7865-7875
Number of pages11
JournalJournal of the American Chemical Society
Volume141
Issue number19
Early online date24 Apr 2019
DOIs
Publication statusPublished - 2019

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Catalytic RNA
Conformations
Nucleophiles
Acids
Metal ions
Catalytic Domain
Substitution reactions
Crystal structure
Metals
Phosphates
Ions
hammerhead ribozyme
Experiments

Keywords

  • RNA catalysis
  • general acid-base catalysis
  • metal ions
  • atomic mutagenesis
  • X-ray crystallography

Cite this

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title = "Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes",
abstract = "Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2′ nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2′-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2′ of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct.",
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note = "We thank Saira Ashraf for expert RNA synthesis, and acknowledge financial support from Cancer Research UK (program grant A18604 to D.M.J.L) and the U.S. National Institutes of Health (GM131568 to J.A.P.). We thank Diamond light source for synchrotron time and the Wellcome Trust for the in-house diffractometer.",
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Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes. / Wilson, Timothy J.; Liu, Yijin; Li, Nan-Sheng; Dai, Qing ; Piccirilli, Joseph A.; Lilley, David M. J. (Lead / Corresponding author).

In: Journal of the American Chemical Society, Vol. 141, No. 19, 2019, p. 7865-7875.

Research output: Contribution to journalArticle

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