Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 A resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner.

Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.

Natriuretic peptides (NPs) are vasoactive cyclic-peptide hormones important in blood pressure regulation through interaction with natriuretic cell-surface receptors. We report the hormone-binding thermodynamics and crystal structures at 2.9 and 2.0 angstroms, respectively, of the extracellular domain of the unliganded human NP receptor (NPR-C) and its complex with CNP, a 22-amino acid NP. A single CNP molecule is bound in the interface of an NPR-C dimer, resulting in asymmetric interactions between the hormone and the symmetrically related receptors. Hormone binding induces a 20 angstrom closure between the membrane-proximal domains of the dimer. In each monomer, the opening of an interdomain cleft, which is tethered together by a linker peptide acting as a molecular spring, is likely a conserved allosteric trigger for intracellular signaling by the natriuretic receptor family.

Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5′ UTR 1 . This discovery has raised the question of whether generegulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes 2,3 are present in the 3′ UTRs of rodent C-type lectin type II (Clec2) genes 4-7 . Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads 8-10 , occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the poly-adenylation signals within the 3′ UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of posttranscriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals.The hammerhead ribozyme is a small, self-cleaving motif composed of a three-helical junction with a core of invariant nucleotides required for activity. To identify hammerhead ribozymes in mammalian mRNAs, we searched mRNA sequence databases using a pattern descriptor that allowed for insertions of up to 5,000 nucleotides at the ends of stem 1 or stem 3 ( Supplementary Fig. 1) 11,12 . Three hammerhead ribozymes were identified in the 3′ UTRs of known rodent mRNAs. Two are found embedded in the transcripts of mouse Clec2d (osteoclast inhibitory lectin, also known as Ocil, Clr-b and Clec2d8) 13 and its paralogue Clec2e (also known as Clra and Clec2d7) 14 , genes that belong to a group of phylogenetically related sequences within the natural killer receptor gene complex of chromosome 6. The third ribozyme is found in rat CLEC2D11 (ref. 7)-a homologue of mouse Clec2d-which resides in the syntenic natural killer receptor gene complex region on chromosome 4. We extended our search to the genomic sequences of other organisms using the UCSC genome browser's comparative genomics tool 15 . Alignments using the natural killer receptor gene complex regions of mouse and rat led to the identification of nine candidate hammerhead ribozymes: four in the 3′ regions of predicted rat, horse and platypus Clec2-like genes, and five in the unannotated regions of five other mammalian genomes ( Supplementary Fig. 2 Unlike most known self-cleaving RNA motifs that are contiguous 8-10,16-19 , the hammerhead ribozymes identified here (referred to as CLEC2 ribozymes) are split into two fragments separated by a long ribozyme-unrelated insertion in the stem-1-capping loop. Th...