Structure-Activity Based Mechanistic Insights into Cleavage Chemistry by Self-Cleaving Nucleolytic Ribozymes

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ABSTRACT Our mechanistic understanding of small (<150 nt) self-cleaving nucleolytic ribozymes that primarily use general- acid base catalysis involving attack of the 2’-OH on the adjacent scissile phosphate to site-specifically cleave the intervening phosphodiester backbone has initially focused on hammerhead, hairpin, glmS, hepatitis delta virus and Varkud Satellite ribozymes, and more recently on twister (Twr), twister-sister (TSr), pistol (Psr) and hatchet (Htr) ribozymes. Our overall goal as reflected from our structure-function studies of the Twr, Tsr, Psr and Htr ribozymes is to expand on our current understanding of the catalytic versatility of RNA with the emphasis on the contributions of active site organization, geometric constraints, activation of the 2’-OH nucleophile, the role of transition state stabilization, protonation of the 5’-oxygen leaving group and the potential of Mg2+ cations in mediating catalysis. One of the challenges in the field relates to whether self-cleaving ribozymes use a common or diverse set of mechanisms, and the extent to which hydrated divalent cations catalyze cleavage chemistry. We have an ongoing collaboration with the Ronald Micura lab (Innsbruck) to study catalytic mechanisms of self- cleaving ribozymes by solving crystal structures of precatalytic, transition and product states in our lab, followed by systematic multi-faceted studies of structure-guided selective catalytic mutants and analogs, as well as pKa measurements of catalytic residues, and pH and temperature-dependence of catalytic rates by the Micura lab. Aim 1: A recent biochemical genome-wide screen resulted in the identification of the naturally occurring self- cleaving hovlinc ribozyme in humans. The sequence of the 168-nt hovlinc ribozyme and its 83-nt minimal functional counterpart contained two pseudoknots with one of them embedded in the cleavage site. The cleavage rate was shown to increase with pH and its inverse correlation with the pKa of divalent cations suggested the catalytic participation of a hydrated divalent cation in cleavage chemistry. We propose to crystallize and determine the structures of the precatalytic, vanadate transition-state mimic and product states of the minimal functional hovlinc ribozyme and follow up with systematic functional studies with the Micura lab of structure- guided modifications and rate measurements towards elucidation of its catalytic cleavage mechanism. Aim 2. This Aim revisits structure-activity relationships of the Twr and TSr ribozymes to resolve discrepancies in the published precatalytic structures and resulting mechanistic insights reported in the literature. The splayed- apart orientation of bases at the cleavage site in a four-way junctional TSr ribozyme by our group contrasts with the stacked bases at the cleavage site in a three-way junctional TSr from the David Lilley lab. We propose to characterize vanadate transition-state mimics of the Twr and TSr ribozymes to resolve the existing discrepancies and mechanistically evaluate the proposed role of catalytic bases and hydrated divalent cations in mediating cleavage chemistry. These studies should elucidate whether Twr and TSr ribozymes, that contain common but topologically distinct conserved sequence elements, use similar or distinct mechanisms for cleavage chemistry.