8 research outputs found
A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination
RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fabâligase complex at 3.1-Ă
resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and FabsâČ high molecular weight, large surface area and phasing power.National Institutes of Health (U.S.) (GM61835
Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme
The Varkud satellite (VS) ribozyme
catalyzes site-specific RNA
cleavage and ligation reactions. Recognition of the substrate involves
a kissing loop interaction between the substrate and the catalytic
domain of the ribozyme, resulting in a rearrangement of the substrate
helix register into a so-called âshiftedâ conformation
that is critical for substrate binding and activation. We report a
3.3 Ă
crystal structure of the complete ribozyme that reveals
the active, shifted conformation of the substrate, docked into the
catalytic domain of the ribozyme. Comparison to previous NMR structures
of isolated, inactive substrates provides a physical description of
substrate remodeling, and implicates roles for tertiary interactions
in catalytic activation of the cleavage loop. Similarities to the
hairpin ribozyme cleavage loop activation suggest general strategies
to enhance fidelity in RNA folding and ribozyme cleavage
Structure-Based Design, Synthesis, and Biological Evaluation of the CageâAmide Derived Orthopox Virus Replication Inhibitors
Despite the fact that the variola virus is considered eradicated, the search for new small molecules with activity against orthopoxviruses remains an important task, especially in the context of recent outbreaks of monkeypox. As a result of this work, a number of amides of benzoic acids containing an adamantane fragment were obtained. Most of the compounds demonstrated activity against vaccinia virus, with a selectivity index SI = 18,214 for the leader compound 18a. The obtained derivatives also demonstrated activity against murine pox (250 †SI †6071) and cowpox (125 †SI †3036). A correlation was obtained between the IC50 meanings and the binding energy to the assumed biological target, the p37 viral protein with R2 = 0.60
Crystal structure of the Varkud satellite ribozyme
Varkud Satellite (VS) ribozyme mediates rolling circle replication of a plasmid found in the Neurospora mitochondria. We report crystal structures of this ribozyme at 3.1Ă
resolution, revealing an intertwined dimer formed by an exchange of substrate helices. Within each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional significance of active site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution