Identification of an imino group indispensable for cleavage by a small ribozyme.
The hairpin ribozyme is a small, noncoding RNA (ncRNA) that catalyzes a site-specific phosphodiester bond cleavage reaction. Prior biochemical and structural analyses pinpointed the amidine moiety of base Ade38 as a key functional group in catalysis, but base changes designed to probe function resulted in localized misfolding of the active site. To define the requirements for chemical activity using a conservative modification, we synthesized and incorporated N1-deazaadenosine into the full-length ribozyme construct. This single-atom variant severely impairs activity, although the active-site fold remains intact in the accompanying crystal structures. The results demonstrate the essentiality of the imino moiety as well as the importance of its interaction with the substrate in the precatalytic and transition-state conformations. This work demonstrates the efficacy of single-atom approaches in the analysis of ncRNA structure-function relationships.
Department of Chemistry, University of Rochester, RC Box 270216, Rochester, New York 14627-0216, USA.
Robert C
RC
Spitale
Rosaria
R
Volpini
Moriah G
MG
Heller
Jolanta
J
Krucinska
Gloria
G
Cristalli
Joseph E
JE
Wedekind
D020134:Catalytic Domain; D007097:Imines / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D007097:Imines; D016337:RNA, Catalytic; C418481:hairpin ribozyme