title: Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch.
abstract: Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3'UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3required five upstream adenylates, and H4a/Ψ3was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3'UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States.
authors.forename: My-Tra
authors.initials: MT
authors.lastname: Le
authors.affiliation: Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, United States.
authors.forename: Wojciech K
authors.initials: WK
authors.lastname: Kasprzak
authors.affiliation: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, United States.
authors.forename: Taejin
authors.initials: T
authors.lastname: Kim
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States.
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States.
authors.forename: Megan Yl
authors.initials: MY
authors.lastname: Young
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Yuan
authors.affiliation: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, United States.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Shapiro
authors.affiliation: Department of Materials Science and Engineering, University of Maryland, College Park, United States.
authors.forename: Joonil
authors.initials: J
authors.lastname: Seog
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, United States.
authors.forename: Anne E
authors.identifier: 0000-0001-6121-0704
authors.initials: AE
authors.lastname: Simon
mesh_terms: D019184:Carmovirus / Q000235:genetics*; D004252:DNA Mutational Analysis; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase
keywords: RNA structure; RNA virus; Turnip crinkle virus; biophysics; molecular dynamics; optical tweezers; structural biology