Local Conformational Changes in the Catalytic Core of the Trans-Acting Hepatitis Delta Virus Ribozyme Accompany Catalysis ² Dinari A. Harris, David Rueda, and Nils G. Walter* Department of Chemistry, The UniVersity of Michigan, 930 North UniVersity, Ann Arbor, Michigan 48109-1055 ReceiVed May 8, 2002; ReVised Manuscript ReceiVed August 15, 2002 ABSTRACT: The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the double- rolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C 75 ) with a pK a close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5′-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G 76 of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP 76 is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP 76 becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C 75 becomes positioned for reaction chemistry only along the trajectory from precursor to product. The hepatitis delta virus ribozyme is among a class of small endonucleolytic RNAs that catalyze a reversible self- cleavage reaction necessary for the replication and propaga- tion of their satellite RNA genomes. Specifically, the hepatitis delta virus ribozyme is a unique RNA motif found in the human hepatitis delta virus (HDV) 1 (1). HDV is a satellite of the hepatitis B virus (HBV); coinfection of HDV and HBV results in intensification of the disease symptoms associated with the hepatitis B virus (2). The small RNA genome of HDV replicates through a double-rolling circle mechanism, whereby multimeric units of genomic and antigenomic RNA strands are produced, followed by self-cleavage and ligation into circular monomers (1, 3). Self-cleavage activity in the genomic and antigenomic RNAs resides within continuous 85-nucleotide sequences that both form a nearly identical secondary structure consisting of a nested double pseudoknot (4, 5). The genomic and antigenomic forms of the HDV ribozyme catalyze self-cleavage by a transesterification reaction, which requires deprotonation of the adjacent 2′-OH group and its nucleophilic attack on the scissile phosphate, resulting in formation of 2′,3′-cyclic phosphate and 5′-OH termini (5). The reaction mechanism of the HDV ribozyme has been extensively studied. The crystal structure of the self-cleaved genomic ribozyme reveals that the base cytosine 75 (C 75 ) is situated in the active site cleft and, thus, in the proximity of the 5′-OH leaving group (Figure 1a,b). Therefore, C 75 in the genomic ribozyme has been proposed to participate directly in reaction chemistry as either a general acid or general base catalyst (6). Several biochemical and mutagenesis studies support the idea that C 75 in the genomic ribozyme and the corresponding RC 76 (R used to distinguish antigenomic numbering) in the antigenomic ribozyme are involved in catalysis (7-10). The pH dependence of self-cleavage (or cis cleavage) by the HDV ribozyme reveals a macroscopic apparent pK a that approaches neutrality. In a widely accepted model, this pK a reflects the ionization equilibrium of N3 in C 75 which therefore is strongly shifted in the folded ribozyme compared to that in the free base (pK a ≈ 4.2). A decrease in this pK a for self- cleavage of an antigenomic ribozyme with an RC 76 A mutation was observed, consistent with A substituting for C in this position to act as a general base catalyst (8). However, the pH profile of the genomic ribozyme in the presence of 1 M NaCl and 1-100 mM EDTA favors a model where C 75 acts as a general acid during catalysis (9, 11). This latter mechanism is in agreement with the crystal structure of the self-cleaved genomic ribozyme, which shows N3 of C 75 within hydrogen bonding distance of the 5′-OH leaving group. In addition, C 75 is hydrogen bonded to the phosphate group of C 22 , increasing the local electron density and providing a mechanism for a shift in pK a , such that it is ² This work was supported by NIH Grant GM62357 to N.G.W., a Rackham Merit predoctoral fellowship and NIH Molecular Biophysics Training Grant to D.A.H., and a postdoctoral fellowship from the Swiss National Fonds to D.R. * To whom correspondence should be addressed. Phone: (734) 615- 2060. Fax: (734) 647-4865. E-mail: nwalter@umich.edu. 1 Abbreviations: AP, 2-aminopurine; FRET, fluorescence resonance energy transfer; fwhm, full width at half-maximum; HDV, hepatitis delta virus. 12051 Biochemistry 2002, 41, 12051-12061 10.1021/bi026101m CCC: $22.00 © 2002 American Chemical Society Published on Web 09/14/2002