Reaction Pathway of the Trans-Acting Hepatitis Delta Virus Ribozyme:  A Conformational Change Accompanies Catalysis

• Published in: Biochemistry (Easton) 2002-01, Vol.41 (3), p.730-740
• Main Authors: Pereira, Miguel J. B, Harris, Dinari A, Rueda, David, Walter, Nils G
• Format: Article
• Language: English
• Subjects: Base Sequence; Calcium - pharmacology; Genome, Viral; Hepatitis D virus; Hepatitis Delta Virus - genetics; Hepatitis Delta Virus - physiology; Humans; Hydrogen-Ion Concentration; Kinetics; Manganese - pharmacology; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligoribonucleotides - chemistry; Phosphorylation; RNA, Catalytic - chemistry; RNA, Catalytic - metabolism; Substrate Specificity; Thermodynamics; Virus Replication
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi011963t

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 × 106 M-1 min-1 at pH 7.5, 11 mM MgCl2, and 25 °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 ∼3 Å) than the free ribozyme, yet becomes significantly extended (by ∼15 Å) 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.