title: Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation.
abstract: Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.
authors.affiliation: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore.
authors.forename: Zhensheng
authors.initials: Z
authors.lastname: Zhong
authors.affiliation: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore.
authors.forename: Lixia
authors.initials: L
authors.lastname: Yang
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore.
authors.forename: Haiping
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore.
authors.forename: Jiahao
authors.initials: J
authors.lastname: Shi
authors.affiliation: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore.
authors.forename: J Jeya
authors.initials: JJ
authors.lastname: Vandana
authors.affiliation: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore.|St Andrew's Junior College, 5 Sorby Adams Drive, 357691 Singapore.
authors.forename: Do Thuy Uyen Ha
authors.initials: DT
authors.lastname: Lam
authors.affiliation: Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: René C L
authors.initials: RC
authors.lastname: Olsthoorn
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore.
authors.forename: Lanyuan
authors.initials: L
authors.lastname: Lu
authors.affiliation: Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore.
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D018965:Frameshifting, Ribosomal; D015683:Gene Products, gag / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D052898:Optical Tweezers; D011489:Protein Denaturation; D017510:Protein Folding; D020365:RNA Helicases / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D015301:Retroviruses, Simian; D012270:Ribosomes / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D015683:Gene Products, gag; D009841:Oligonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D020365:RNA Helicases