Spinach is an in vitro selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence.Spinach is thus an RNA analog of GFP, and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2 and 2.4 Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially pre-formed binding site for the fluorophore.The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

SUMMARYIn nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a multi-megadalton machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, following the discovery of self-splicing group II intron RNAs, the snRNAs were hypothesized to catalyze splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported to date. By using metal rescue strategies, here we show that the U6 snRNA catalyzes both splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Strikingly, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms, and likely common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome.

Summary CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 Å resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-β1-phosphomycoketide (MPM). CD1c accommodated MPM’s methylated alkyl chain exclusively in the A′ pocket, aided by a unique exit portal underneath the α1 helix. Most striking was an open F′ pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical Major Histocompatibility Complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

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.

Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK a ¼ 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolatemodified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756. 5′-phosphorothiolate | RNA catalysis | nucleolytic ribozymes | catalytic mechanism R ibozyme-mediated catalysis is important for both RNA splicing and translation (1), yet its chemical origins are incompletely understood. The nucleolytic ribozymes bring about the site-specific cleavage or ligation of RNA, with an acceleration of a millionfold or greater. The intensively studied protein RNase A catalyses an identical cleavage reaction, and much evidence supports the hypothesis that each of these phosphoryl transfer reactions is subject to general acid-base catalysis. This mechanism requires a general base to deprotonate the attacking nucleophile, and a general acid to protonate the oxyanion leaving group (Fig. 1).The most common chemical entities implicated in RNA catalysis by the nucleolytic ribozymes are the nucleobases (2). Guanine appears to play a catalytic role in the hairpin, hammerhead, GlmS, and Varkud satellite (VS) ribozymes, adenine in the hairpin, and VS and cytosine in the hepatitis delta virus (HDV) ribozyme. Crystal structures of the hairpin ribozyme (3) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, where they seem poised to act in general acid-base catalysis. This is consistent with the pH dependence of the reaction (4) and its variation with functional group modifications (5-8).In its simplest active form, the VS ribozyme comprises five helices (II through VI) organized by two three-way junctions, which acts in trans upon a substrate stem loop (helix I) with an internal loop that contains the scissile phosphate (Fig. 1). The loop also contains the critical G638 (9). A756 (10-13) is contained within an internal loop in helix VI. While no crystal structure of the VS ribozyme has yet been solved, a small-angle X-ray scattering-derived model plac...