The Ribosome Uses Two Active Mechanisms to Unwind mRNA During Translation

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.

[1]  H. Stark,et al.  GTPase Mechanisms and Functions of Translation Factors on the Ribosome , 2000, Biological chemistry.

[2]  K. Shokat,et al.  Human Catechol-O-Methyltransferase Haplotypes Modulate Protein Expression by Altering mRNA Secondary Structure , 2006, Science.

[3]  Joachim Frank,et al.  Locking and Unlocking of Ribosomal Motions , 2003, Cell.

[4]  X. Xie,et al.  Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates. , 2010 .

[5]  J. Holton,et al.  Structures of the Bacterial Ribosome at 3.5 Å Resolution , 2005, Science.

[6]  D. Andrews,et al.  The signal recognition particle receptor alpha subunit assembles co‐translationally on the endoplasmic reticulum membrane during an mRNA‐encoded translation pause in vitro. , 1996, The EMBO journal.

[7]  Harry F. Noller,et al.  The Path of Messenger RNA through the Ribosome , 2001, Cell.

[8]  Z. Tsuchihashi,et al.  Translational frameshifting in the Escherichia coli dnaX gene in vitro. , 1991, Nucleic acids research.

[9]  Ignacio Tinoco,et al.  The effect of force on thermodynamics and kinetics of single molecule reactions. , 2002, Biophysical chemistry.

[10]  Marina V. Rodnina,et al.  Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy , 2010, Nature.

[11]  H. Noller,et al.  mRNA Helicase Activity of the Ribosome , 2005, Cell.

[12]  N. Saitou,et al.  Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. , 2003, Human molecular genetics.

[13]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[14]  T. Lohman,et al.  Non-hexameric DNA helicases and translocases: mechanisms and regulation , 2008, Nature Reviews Molecular Cell Biology.

[15]  M. Betterton,et al.  Opening of nucleic-acid double strands by helicases: active versus passive opening. , 2005, Physical review. E, Statistical, nonlinear, and soft matter physics.

[16]  Harry F. Noller,et al.  Intermediate states in the movement of transfer RNA in the ribosome , 1989, Nature.

[17]  Smita S. Patel,et al.  Mechanisms of Helicases* , 2006, Journal of Biological Chemistry.

[18]  Kristen K. Dang,et al.  Architecture and Secondary Structure of an Entire HIV-1 RNA Genome , 2009, Nature.

[19]  V. Croquette,et al.  Active and passive mechanisms of helicases , 2010, Nucleic acids research.

[20]  M. Rodnina,et al.  Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome. , 2000, Molecular cell.

[21]  Daniel R Southworth,et al.  Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex. , 2003, Molecular cell.

[22]  S. Benkovic,et al.  Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism , 2007, Proceedings of the National Academy of Sciences.

[23]  Sotaro Uemura,et al.  Peptide bond formation destabilizes Shine–Dalgarno interaction on the ribosome , 2007, Nature.

[24]  C. Bustamante,et al.  The mechanochemistry of molecular motors. , 2000, Biophysical journal.

[25]  Ignacio Tinoco,et al.  Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of –1 ribosomal frameshifting , 2009, Proceedings of the National Academy of Sciences.

[26]  N. Oppenheimer,et al.  Structure and mechanism , 1989 .

[27]  Michelle D. Wang,et al.  Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase , 2007, Cell.

[28]  Ignacio Tinoco,et al.  Following translation by single ribosomes one codon at a time , 2008, Nature.

[29]  D. Giedroc,et al.  Frameshifting RNA pseudoknots: Structure and mechanism , 2008, Virus Research.

[30]  I. Tinoco,et al.  Simulation and analysis of single-ribosome translation , 2009, Physical biology.

[31]  Joachim Frank,et al.  A ratchet-like inter-subunit reorganization of the ribosome during translocation , 2000, Nature.