Reaction pathway of the trans-acting hepatitis delta virus ribozyme: a conformational change accompanies catalysis
| Autor: | Miguel J. B. Pereira, David Rueda, Nils G. Walter, Dinari A. Harris |
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| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular Conformational change Stereochemistry Molecular Sequence Data Genome Viral Virus Replication Biochemistry Virus Substrate Specificity Reaction rate constant Circular RNA Humans RNA Catalytic Phosphorylation Manganese Oligoribonucleotides biology Base Sequence Chemistry Ribozyme Hydrogen-Ion Concentration Crystallography Kinetics Förster resonance energy transfer Viral replication biology.protein Nucleic Acid Conformation Thermodynamics Calcium Hepatitis Delta Virus Hairpin ribozyme |
| Zdroj: | Biochemistry. 41(3) |
| ISSN: | 0006-2960 |
| Popis: | The hepatitis delta virus (HDV), an infectious human pathogen and satellite of hepatitis B virus, leads to intensified disease symptoms, including progression to liver cirrhosis. Both the circular RNA genome of HDV and its complementary antigenome contain the same cis-cleaving catalytic RNA motif that plays a crucial role in virus replication. Previously, the high-resolution crystal structure of the product form of a cis-acting genomic HDV ribozyme has been determined, while a trans-acting version of the ribozyme was used to dissect the cleavage reaction pathway. Using fluorescence resonance energy transfer (FRET) on a synthetic trans-cleaving form of the ribozyme, we are able to directly observe substrate binding (at a rate constant k(on) of 7.8 x 10(6) M(-1) min(-1) at pH 7.5, 11 mM MgCl(2), and 25 degrees C) and dissociation (at 0.34 min(-1)). Steady-state and time-resolved FRET experiments in solution and in nondenaturing gels reveal that the substrate (precursor) complex is slightly more compact (by approximately 3 A) than the free ribozyme, yet becomes significantly extended (by approximately 15 A) upon cleavage and product complex formation. We also find that trans cleavage is characterized by a high transition-state entropy (-26 eu). We propose that the significant global conformational change that we observe between the precursor and product structures occurs on the reaction trajectory into a constrained product complex-like transition state. Our observations may present the structural basis of the recently described utilization of intrinsic substrate binding energy to the overall catalytic rate enhancement by the trans-acting HDV ribozyme. |
| Databáze: | OpenAIRE |
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