Talk abstracts

Talk on Saturday 12:30-12:50pm submitted by Barbara Golden

A 1.9 Å crystal structure of the HDV ribozyme precleavage suggest both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage

Jui-Hui Chen (Department of Biochemistry, Purdue University), Rieko Yajima (Department of Chemistry, The Pennsylvania State University), Durga M. Chadalavada (Department of Chemistry, The Pennsylvania State University), Elaine Chase (Department of Biochemistry, Purdue University), Philip C. Bevilacqua (Department of Chemistry, The Pennsylvania State University), Barbara L. Golden (Department of Biochemistry, Purdue University)

Abstract:
The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pK_a shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg²⁺ ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg²⁺ ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg²⁺ ion is also found within the active site where it interacts with a newly resolved G•U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-R_P oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are innersphere ligands to the active site Mg²⁺ ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

Keywords: ribozyme, mechanism, HDV