Mechanistic characterization of the HDV genomic ribozyme: The cleavage site base pair plays a structural role in facilitating catalysis

Andrea L. Cerrone-Szakal; Durga M. Chadalavada; Barbara L. Golden; Philip C. Bevilacqua

doi:10.1261/rna.1140308

Mechanistic characterization of the HDV genomic ribozyme: The cleavage site base pair plays a structural role in facilitating catalysis

¹Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
²Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA

↵3 Present Address: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850, USA.

Abstract

The hepatitis delta virus (HDV) ribozyme occurs in the genomic and antigenomic strands of the HDV RNA and within mammalian transcriptomes. Previous kinetic studies suggested that a wobble pair (G•U or A⁺•C) is preferred at the cleavage site; however, the reasons for this are unclear. We conducted sequence comparisons, which indicated that while G•U is the most prevalent combination at the cleavage site, G-C occurs to a significant extent in genomic HDV isolates, and G•U, G-C, and A-U pairs are present in mammalian ribozymes. We analyzed the folding of genomic HDV ribozymes by free energy minimization and found that variants with purine–pyrimidine combinations at the cleavage site are predicted to form native structures while pyrimidine–purine combinations misfold, consistent with earlier kinetic data and sequence comparisons. To test whether the cleavage site base pair contributes to catalysis, we characterized the pH and Mg²⁺-dependence of reaction kinetics of fast-folding genomic HDV ribozymes with cleavage site base pair purine–pyrimidine combinations: G•U, A-U, G-C, and A⁺•C. Rates for these native-folding ribozymes displayed highly similar pH and Mg²⁺ concentration dependencies, with the exception of the A⁺•C ribozyme, which deviated at high pH. None of the four ribozymes underwent miscleavage. These observations support the A⁺•C ribozyme as being more active with a wobble pair at the cleavage site than with no base pair at all. Overall, the data support a model in which the cleavage site base pair provides a structural role in catalysis and does not need to be a wobble pair.

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Footnotes

Reprint requests to: Philip C. Bevilacqua, Department of Chemistry, 104 Chemistry Bldg., The Pennsylvania State University, University Park, PA 16802, USA; e-mail: pcb{at}chem.psu.edu; fax: (814) 863-8403.
↵1 As a shorthand, we refer to the cleavage site base pair as bp 1.
↵2 The small (2 bp) P1.1 pairing is not predicted correctly. Instead, formation of a base pair between U- 1 and G38, which extends P1 by 1 bp, is predicted. In addition, the base pair between A43 and G74 at the top of P4 is not predicted. Lastly, a terminal base pair in L4 between A56 and U60 is predicted, while experiments coupled with mFold predictions suggest that these bases are single stranded at least part of the time (Chadalavada et al. 2000).
↵3 Previously described alternative pairings are denoted with (previously assigned) numbering (e.g., Alt 1), while new alternative pairings are denoted with lettering (e.g., Alt A).
↵4 The AC ribozyme k_max/K_d^{α H} value is higher because both K_d and k_max are two- to threefold lower (Table 3) and the ribozyme may bind an additional Mg²⁺ ion. It is therefore not straightforward to compare the k_max/K_d^{α H} value for the AC ribozyme at higher pH.
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1140308.
- Received April 16, 2008.
- Accepted May 30, 2008.