Characterizing the Dynamic Tertiary Interaction that Promotes Cleavage in an Extended Hammerhead Ribozyme (2015)

Undergraduates: Quoc Mac, Bo Zhao

Faculty Advisor: Qi Zhang
Department: Chemistry

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Recent bioinformatics studies have revealed the ubiquitous presence of hammerhead ribozyme, a small self-cleaving RNA, in all domains of life, from bacteria to complex eukaryotes. Their fundamental catalytic activity as well as genomic locations suggest some important biological functions, especially in gene regulation. While a minimal hammerhead catalytic core is essential for cleavage activity, the presence of transient tertiary interactions between stem I and stem II of the extended hammerhead helps to enhance cleavage by 1000 fold. In this study, we attempted to characterize the tentative tertiary interaction of c10orf118 hammerhead ribozyme, an ultra-conserved hammerhead found in mammalian species. While it is suggested that a pseudoknot interaction helps to bring the two stems together, we identified that this interaction is in fact facilitated by several base pairs within the bulge of stem I and the loop of stem II. By using CEST, a newly developed NMR dynamic experiment, we were able to detect the presence of a lowly-populated excited state thought to be the active state of the ribozyme. A mutant that carries a UUCG loop at stem II, surprisingly, not only reduces the cleavage rate, but almost completely abolishes the catalytic activity. From this evidence, we believe that not only the identities of residues in the loop-bulge region but also the relative orientation of the two stems are fundamental for cleavage enhancement.