title: Single-molecule measurements of the CCR5 mRNA unfolding pathways.
abstract: Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a -1 programmed ribosomal frameshift (-1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human -1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the -1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism.
authors.affiliation: Department of Physics, Institute for Physical Science and Technology Biophysics Program, University of Maryland, College Park, Maryland.
authors.forename: Michel
authors.initials: M
authors.lastname: de Messieres
authors.affiliation: Department of Physics, Institute for Physical Science and Technology Biophysics Program, University of Maryland, College Park, Maryland.
authors.forename: Jen-Chien
authors.initials: JC
authors.lastname: Chang
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland.
authors.forename: Ashton Trey
authors.initials: AT
authors.lastname: Belew
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland.
authors.forename: Arturas
authors.initials: A
authors.lastname: Meskauskas
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Dinman
authors.affiliation: Department of Physics, Institute for Physical Science and Technology Biophysics Program, University of Maryland, College Park, Maryland. Electronic address: alaporta@umd.edu.
authors.forename: Arthur
authors.initials: A
authors.lastname: La Porta
mesh_terms: D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D059372:Nucleotide Motifs; D052898:Optical Tweezers; D059370:RNA Folding; D012333:RNA, Messenger / Q000737:chemistry*; D019713:Receptors, CCR5 / Q000235:genetics*; D013816:Thermodynamics
chemical_list: C584933:CCR5 protein, human; D012333:RNA, Messenger; D019713:Receptors, CCR5