Dataset Information

Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes.

ABSTRACT: Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2' nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2'-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2' of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct.

SUBMITTER: Wilson TJ

PROVIDER: S-EPMC6523996 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes.

Wilson Timothy J TJ Liu Yijin Y Li Nan-Sheng NS Dai Qing Q Piccirilli Joseph A JA Lilley David M J DMJ

Journal of the American Chemical Society 20190502 19

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2' nucleophile, and the conformation at the scissile phosphate and the position of the gene ...[more]

PMID: 31017785

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Project description:BackgroundRibozymes are small catalytic RNAs that possess the dual functions of sequence-specific RNA recognition and site-specific cleavage. Trans-cleaving ribozymes can inhibit translation of genes at the messenger RNA (mRNA) level in both eukaryotic and prokaryotic systems and are thus useful tools for studies of gene function. However, identification of target sites for efficient cleavage poses a challenge. Here, we have considered a number of structural and thermodynamic parameters that can affect the efficiency of target cleavage, in an attempt to identify rules for the selection of functional ribozymes.ResultsWe employed the Sfold program for RNA secondary structure prediction, to account for the likely population of target structures that co-exist in dynamic equilibrium for a specific mRNA molecule. We designed and prepared 15 hammerhead ribozymes to target GUC cleavage sites in the mRNA of the breast cancer resistance protein (BCRP). These ribozymes were tested, and their catalytic activities were measured in vitro. We found that target disruption energy owing to the alteration of the local target structure necessary for ribozyme binding, and the total energy change of the ribozyme-target hybridization, are two significant parameters for prediction of ribozyme activity. Importantly, target disruption energy is the major contributor to the predictability of ribozyme activity by the total energy change. Furthermore, for a target-site specific ribozyme, incorrect folding of the catalytic core, or interactions involving the two binding arms and the end sequences of the catalytic core, can have detrimental effects on ribozyme activity.ConclusionThe findings from this study suggest rules for structure-based rational design of trans-cleaving hammerhead ribozymes in gene knockdown studies. Tools implementing these rules are available from the Sribo module and the Srna module of the Sfold program available through Web server at http://sfold.wadsworth.org.

Dataset Information

Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes.

Publications

Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes.

Similar Datasets

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