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      Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape.

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          Abstract

          The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2'-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape.

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          Author and article information

          Journal
          RNA
          RNA (New York, N.Y.)
          Cold Spring Harbor Laboratory
          1469-9001
          1355-8382
          Jul 2014
          : 20
          : 7
          Affiliations
          [1 ] Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
          [2 ] Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
          [3 ] Regional Centre of Advance Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University, 771 46 Olomouc, Czech Republic.
          [4 ] Institute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Brno, Czech Republic Masaryk University, Campus Bohunice, 625 00 Brno, Czech Republic.
          [5 ] Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
          Article
          rna.044982.114
          10.1261/rna.044982.114
          4114689
          24854621

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