Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity

  title={Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity},
  author={Anastasia Khvorova and Aur{\'e}lie Lescoute and Eric Westhof and Sumedha D. Jayasena},
  journal={Nature Structural Biology},
The hammerhead ribozyme (HHRz) is a small, naturally occurring ribozyme that site-specifically cleaves RNA and has long been considered a potentially useful tool for gene silencing. The minimal conserved HHRz motif derived from natural sequences consists of three helices that intersect at a highly conserved catalytic core of 11 nucleotides. The presence of this motif is sufficient to support cleavage at high Mg2+ concentrations, but not at the low Mg2+ concentrations characteristic of… 
The hammerhead ribozyme: structure, catalysis, and gene regulation.
Intracellular selection of trans-cleaving hammerhead ribozymes
Three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme were obtained and the best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead Ribozyme.
Characterization of a native hammerhead ribozyme derived from schistosomes.
A kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate, and loop-loop interactions do aid in forming a more active conformation.
Restoration of Ribozyme Tertiary Contact and Function by Using a Molecular Glue for RNA.
It is demonstrated that the rationally designed ribozyme switch enabled regulation of gene expression by Z-NCTS and was functional in mammalian cells.
Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis
Hammerhead Ribozymes Against Virus and Viroid RNAs
The hammerhead ribozyme, a small catalytic motif that promotes self-cleavage of the RNAs in which it is found naturally embedded, can be manipulated to recognize and cleave specifically in trans
A database search for hammerhead ribozyme motifs.
A database search for novel examples of this catalytic RNA, taking into consideration the recently defined structural requirements for an efficient cleavage under physiological magnesium ion concentrations, finds several hundreds of motifs in organisms of all kingdoms of life.
Functional Analysis of Hairpin Ribozyme Active Site Architecture*
This study focuses on the network of noncovalent interactions that align nucleophilic and leaving group oxygens in the orientation required for the SN2-type reaction mechanism and orient the active site nucleobases near the reactive phosphate to facilitate catalytic chemistry.
Probing general acid catalysis in the hammerhead ribozyme.
A functional characterization of the general acid catalysis mechanism and the role of an M(2+) cofactor therein, for the S. mansoni hammerhead and a simple method for synthesizing radiolabeled bridging-5'-phosphorothioate substrates are reported.
The structure-function dilemma of the hammerhead ribozyme.
This work has evaluated and distilled the large body of biochemical data into a consensus set of functional groups unambiguously required for hammerhead catalysis, and established a concise set of disagreements between the structural and functional data.


Sequence requirements of the hammerhead RNA self-cleavage reaction.
The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA.
Three-dimensional structure of a hammerhead ribozyme
The X-ray crystallographic structure of a hammerhead RNA–DNA ribozyme-inhibitor complex at 2.6 Å resolution reveals that the base-paired stems are A-form helices and the core has two structural domains.
Hammerhead ribozyme kinetics.
The X-ray crystal structures of two hammerhead ribozyme–inhibitor complexes revealed that the core residues fold into two separate domains and the helices are arranged in a Y-shape conformation.
Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions.
  • D. M. Long, O. Uhlenbeck
  • Biology, Chemistry
    Proceedings of the National Academy of Sciences of the United States of America
  • 1994
It is confirmed that both reactions probably have the same rate-limiting step, and the relative activities of three sequences selected from the intramolecular library are the same when the sequences are transferred into an intermolecular hammerhead background.
Kinetic Mechanism of the Hairpin Ribozyme
Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates, which can be explained by stabilization of the catalytic core of the ribozyme.
Tertiary structure stabilization promotes hairpin ribozyme ligation.
The enhanced ligation activity ofhairpin ribozymes that contain a four-way helical junction supports the notion that tertiary structure stability is a major determinant of the hairpin ribozyme proficiency as a ligase and illustrates the link between RNA structure and biological function.
The folding of the hairpin ribozyme: dependence on the loops and the junction.
The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form ofThe ribo enzyme.