Structural Basis for Fluorescence Activation by Pepper RNA
Pepper is a fluorogenic RNA aptamer tag that binds to a variety of benzylidene-cyanophenyl (HBC) derivatives with tight affinity and activates their fluorescence. To investigate how Pepper RNA folds to create a binding site for HBC, we used antibody-assisted crystallography to determine the structures of Pepper bound to HBC530 and HBC599 to 2.3 and 2.7 Å resolutions, respectively. The structural data show that Pepper folds into an elongated structure and organizes nucleotides within an internal bulge to create the ligand binding site, assisted by an out-of-plane platform created by tertiary interactions with an adjacent bulge. As predicted from a lack of K+ dependence, Pepper does not use a G-quadruplex to form a binding pocket for HBC. Instead, Pepper uses a unique base-quadruple·base-triple stack to sandwich the ligand with a U·G wobble pair. Site-bound Mg2+ ions support ligand binding structurally and energetically. This research provides insight into the structural features that allow the Pepper aptamer to bind HBC and show how Pepper’s function may expand to allow the in vivo detection of other small molecules and metals.
In January 2022 our group published a new paper in Nature Chemical Biology, where we describe a first crystal structure of a self-alkylating ribozyme along with some insights on the mechanism of reaction.
Structural basis for substrate binding and catalysis by a self-alkylating ribozyme
Ribozymes that react with small-molecule probes have important applications in transcriptomics and chemical biology, such as RNA labeling and imaging. Understanding the structural basis for these RNA-modifying reactions will enable the development of better tools for studying RNA. Nevertheless, high-resolution structures and underlying catalytic mechanisms for members of this ribozyme class remain elusive. Here, we focus on a self-alkylating ribozyme that catalyzes nitrogen–carbon bond formation between a specific guanine and a 2,3-disubstituted epoxide substrate and report the crystal structures of a self-alkylating ribozyme, including both alkylated and apo forms, at 1.71-Å and 2.49-Å resolution, respectively. The ribozyme assumes an elongated hairpin-like architecture preorganized to accommodate the epoxide substrate in a hook-shaped conformation. Observed reactivity of substrate analogs together with an inverse, log-linear pH dependence of the reaction rate suggests a requirement for epoxide protonation, possibly assisted by the ether oxygens within the substrate.
The programmed −1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well-conserved structured RNA found in all coronaviruses’ genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in a continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1−10 and NSPs 1−16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the −1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the −1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three- dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.
Picornaviral IRES elements are essential for initiating the cap-independent viral translation. However, three-dimensional structures of these elements remain elusive. Here, we report a 2.84-Å resolution crystal structure of hepatitis A virus IRES domain V (dV) in complex with a synthetic antibody fragment—a crystallization chaperone. The RNA adopts a three-way junction structure, topologically organized by an adenine-rich stem-loop motif. Despite no obvious sequence homology, the dV architecture shows a striking similarity to a circularly permuted form of encephalomyocarditis virus J-K domain, suggesting a conserved strategy for organizing the domain architecture. Recurrence of the motif led us to use homology modeling tools to compute a 3-dimensional structure of the corresponding domain of foot-and-mouth disease virus, revealing an analogous domain organizing motif. The topological conservation observed among these IRESs and other viral domains implicates a structured three-way junction as an architectural scaffold to pre-organize helical domains for recruiting the translation initiation machinery.