Reaction pathway of the trans-acting hepatitis delta virus ribozyme: a conformational change accompanies catalysis.
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.
Pereira
Miguel J B
MJ
Department of Chemistry, The University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, USA.
Harris
Dinari A
DA
Rueda
David
D
Walter
Nils G
NG
D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D016679:Genome, Viral; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry; D010766:Phosphorylation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014779:Virus Replication
D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D008345:Manganese; D002118:Calcium