Karolina came to us from Polish Academy of Science in Warsaw, Poland as a part of Fulbright’s BioLAB program and will stay with us for a year! Karolina will spend her internship on structural characterization of RNA-antibody complexes to gain better understanding on what structural features antibodies adopt to promote effective RNA binding. Welcome!
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.
https://www.nature.com/articles/s41467-019-11585-z
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.
Our group is broadly interested in the chemistry and biochemistry of nucleic acids with particular emphasis on RNA and RNA catalysis. The laboratory integrates areas of organic chemistry, physical chemistry, enzymology and molecular biology to gain a fundamental understanding of nucleic acid structure and mechanisms of RNA catalysis. Using the principles and techniques of organic chemistry and molecular biology, we manipulate the structure of RNA molecules at precise locations in ways that are designed to answer very specific questions about biological function. With a team consisting of people trained in EPR, Optical Tweezers, Fluorescence spectroscopy, MRI and X-Ray Crystallography, protein engineering and Nucleic acid synthetic chemistry, we are equipped to take on varied challenges in RNA chemistry and biology.
Apply for Postdoc, Graduate and Undergraduate Positions
Please send a resume and a description of research accomplishments, research interests, and long-term goals. All inquires should be emailed to: jpicciri@uchicago.edu