A 1.9 Å Crystal Structure of the HDV Ribozyme Precleavage Suggests both Lewis Acid and General Acid Mechanisms Contribute to Phosphodiester Cleavage

Journal Article · Mon Nov 01 04:00:00 EDT 2010 · Biochemistry-US

DOI:https://doi.org/10.1021/bi100670p· OSTI ID:1002758

Chen, Jui-Hui; Yajima, Rieko; Chadalavada, Durga M; Chase, Elaine; Bevilacqua, Philip C; Golden, Barbara L ^[1]

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1. Purdue

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{prime},3{prime}-cyclic phosphate and 5{prime}-hydroxyl termini. The active site nucleotide C75 has a pK{sub a} shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg{sup 2+} ion that helps activate the 2{prime}-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 Mg{sup 2+} 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 Mg{sup 2+} ion is also found within the active site where it interacts with a newly resolved G {center_dot} 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-RP oxygen of the scissile phosphate and the 2{prime}-hydroxyl nucleophile are inner-sphere ligands to the active site Mg{sup 2+} ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

• Research Organization: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
• Sponsoring Organization: USDOE
• OSTI ID: 1002758
• Journal Information: Biochemistry-US, Journal Name: Biochemistry-US Journal Issue: (31) ; 08, 2010 Vol. 49; ISSN 0006-2960; ISSN 1520-4995; ISSN BICHAW
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

36 MATERIALS SCIENCE
CATALYSIS
CLEAVAGE
CRYSTAL STRUCTURE
HEPATITIS
LEWIS ACIDS
NUCLEOTIDES
OXYGEN
PHOSPHATES
RNA