Author Archives: hcrees

The SARS-CoV‑2 Programmed −1 Ribosomal Frameshifting Element Crystal Structure Solved to 2.09 Å Using Chaperone-Assisted RNA Crystallography

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.

3D RNA structures for recruiting translation machinery

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.

Welcome to the Piccirilli Lab

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.

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