Triplex Structures in an RNA Pseudoknot Enhance Mechanical Stability and Increase Efficiency of 1 Ribosomal Frameshifting
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1 Triplex Structures in an RNA Pseudoknot Enhance Mechanical Stability and Increase Efficiency of 1 Ribosomal Frameshifting Gang CHEN Department of Molecular Biology The Scripps Research Institute 5 October, 2009
2 One amino acid in a protein is coded by three nucleotides in a messenger RNA 0 frame: A U G U A G Start codon Stop codon
3 Ribosomal reading frames +1 frame: A U G U A G 0 frame: A U G U A G 1 frame: A U G U A G Intrinsic frameshift error: <5 10 5
4 Structures in mrna coding regions have to be unfolded for translation 5' Stem 2 3' Loop 2 5' Stem 1 Stem 2 3' Loop 1 5' 3' Hairpin Pseudoknot (2D) Pseudoknot (3D) Puglisi, Wyatt, and Tinoco, (1988) Nature
5 Structure of prokaryotic ribosome Korostelev, Ermolenko, and Noller (2008) Curr. Opin. Chem. Biol.
6 Three components for programmed 1 ribosomal frameshifting slippery sequence single strand spacer (5-10 nucleotides) pseudoknot 0 frame: start codon X XXY YYZ 1 frame: start codon XXX YYY Z X is any nucleotide, Y is A or U, and Z is usually not G
7 Pseudoknot structure stimulates 1 ribosomal frameshifting Drawing is not to scale
8 Programmed 1 ribosomal frameshifting is utilized by many viruses Human immunodeficiency virus (HIV) ( 1 frameshifting efficiency: <10%). Severe Acute Respiratory Syndrome coronavirus (SARS- Cov) ( 1 frameshifting efficiency: ~15%). Novel anti-virus therapy may be developed by targeting 1 frameshifting.
9 Choosing a model pseudoknot to study 1 ribosomal frameshifting High resolution three dimensional structure High efficiency (>40%) in stimulating 1 frameshifting
10 NMR structure of human telomerase RNA pseudoknot ( U177) Theimer, Blois, and Feigon, (2005) Mol. Cell
11 Base triples in human telomerase RNA pseudoknot ( U177) Minor groove base triples Major groove base triples Theimer, Blois, and Feigon, (2005) Mol. Cell
12 Major groove base triples U. A-U C +. G-C
13 Human telomerase RNA pseudoknot ( U177) as a model system to study 1 frameshifting Are pseudoknot base triples required for high efficiency 1 ribosomal frameshifting? How do base triples affect mechanical stability of RNA How do base triples affect mechanical stability of RNA pseudoknot?
14 The construct for bulk frameshifting assay start codon In vitro translation in rabbit reticulocyte lysate (Promega) 0 frame stop codon
15 The construct for bulk frameshifting assay start codon 0 frame: Smaller protein 1 frame: Larger fusion protein In vitro translation in rabbit reticulocyte lysate (Promega) 0 frame stop codon 1 frame stop codon
16 Pseudoknot mutants based on U177: Mutations in loops C C C G U Pseudoknot Number of base triples disrupted U177 0 TeloWT (with U177) 0 100C115C174G 0 101C114C175G 0 115C174G 1 114C175G 1 101C 1 102C 1 GU 2 CCC 3 CCCGU 5
17 Pseudoknot mutants based on U177: Mutations in loops 101C 102C Pseudoknot Number of base triples disrupted U177 0 TeloWT (with U177) 0 100C115C174G 0 101C114C175G 0 115C174G 1 114C175G 1 101C 1 102C 1 GU 2 CCC 3 CCCGU 5
18 Pseudoknot mutants based on U177: Mutations in stem 2 115C174G 114C175G Pseudoknot Number of base triples disrupted U177 0 TeloWT (with U177) 0 100C115C174G 0 101C114C175G 0 115C174G 1 114C175G 1 101C 1 102C 1 GU 2 CCC 3 CCCGU 5
19 Pseudoknot mutants based on U177: Mutations in stem 2 and loop 1 Pseudoknot Number of base triples disrupted U177 0 TeloWT (with U177) 0 100C115C174G 0 100C115C174G 101C114C175G 0 101C114C175G 115C174G 1 114C175G 1 101C 1 102C 1 GU 2 CCC 3 CCCGU 5
20 Pseudoknot mutants based on U177: Mutations in stem 2 U177 bulge Pseudoknot Number of base triples disrupted U177 0 TeloWT (with U177) 0 100C115C174G 0 101C114C175G 0 115C174G 1 114C175G 1 101C 1 102C 1 GU 2 CCC 3 CCCGU 5
21 Frameshifting efficiency measured by SDS-PAGE CCCGU CCC GU C174G 114C175G 102C 100C115C174G 101C114C175G U Number of base triples disrupted frame product 0 frame product 1 frameshifting efficiency (%) Pseudoknot base triples are required for high-efficiency 1 frameshifting
22 Pseudoknot base triples are required for high efficiency 1 frameshifting # of base triples disrupted: 0 1 > iency (%) C C C G U frameshift effici Delta U C114C175G 100C115C174G TeloWT 102C 114C175G 115C174G 101C GU CCC CCCGU U C175G 115C174G 101C GU CCC CCCGU 101C 102C 100C115C174G 115C174G 114C175G 101C114C175G U177 bulge Pseudoknot construct 101C114C175G 100C115C174G TeloWT 102C
23 Bulk studies: Pseudoknot base triples are required for high efficiency (>40%) 1 ribosomal frameshifting. Single-molecule studies: How does RNA pseudoknot unfold and fold? How do base triples affect mechanical stability of pseudoknot? Is there correlation between pseudoknot mechanical stability and 1 ribosomal frameshifting efficiency?
24 Bulk thermal unfolding pathway Stem 1 hairpin Folding intermediate Native pseudoknot High temperature Medium temperature Low temperature Theimer, Blois, and Feigon, (2005) Mol. Cell
25 Optical trap behaves as a linear Hooke spring F = k X 1 kcal/mol corresponds to ~7 pn nm or 1.7 k B T (298 K) Moffitt, Chemla, Smith, and Bustamante, (2008) Annu. Rev. Biochem.
26 Single-molecule mechanical (un)folding of RNA Mechanical force is a unique physical variable which is applied locally to RNA structures. RNA folding and unfolding can be studied in near physiological solution and at room temperature. Kinetic intermediates can be directly revealed.
27 Optical tweezers for single-molecule manipulation of RNA pseudoknots Force Force 5 5 Streptavidin-coated pipette bead 592-bp RNA/DNA handle RNA pseudoknot 543-bp RNA/DNA handle 3 3 Anti-digoxigenin-coated trap bead Force Force 200 mm NaCl, 10 mm Tris-HCl, 0.1 mm EDTA, ph 7.3, 22 ºC
28 Three classes of unfolding trajectories 50 Unfolding force Extension increase 40 Force (pn) a b c d e f g Hop 10 h Unfolding force (pn) U177 The second step of 2-step unfolding High-force 1-step unfolding Low-force 1-step unfolding Extension change (nm) Pulling rate: 100 nm/s Extension (nm) 60 i Unfolding force (pn) The second step of 2-step unfolding Mutant CCC 40 nm High-force 1-step unfolding Low-force 1-step unfolding Extension change (nm) m
29 Three classes of unfolding trajectories 50 Unfolding force Extension increase CCC mutant 40 Force (pn) a b c d e f g Hop C C C 10 h Unfolding force (pn) U177 The second step of 2-step unfolding High-force 1-step unfolding Low-force 1-step unfolding Extension change (nm) Pulling rate: 100 nm/s Extension (nm) 60 i Unfolding force (pn) CCC Mutant mutant CCC The second step of 2-step unfolding 40 nm High-force 1-step unfolding Low-force 1-step unfolding Extension change (nm) m
30 NMR structure of stem 1 hairpin Theimer, Finger, Trantirek, and Feigon, (2003) Proc. Natl. Acad. Sci. U.S.A.
31 Isolated stem 1 and stem 2 hairpins for singlemolecule studies 5' 3' 5' 3' Isolated stem 1 hairpin (29 nt) Isolated stem 2 hairpin (30 nt)
32 Analysis of the second step of two-step unfolding forces Low force two-step reaction (2 nd step) Unfolding force (pn) Delta U177 U Extension change (nm) High force one-step reaction Low force one-step reaction Which stem is disrupted during the second step of low force two-step unfolding?
33 Unfolding force comparison Force (pn) U177 Stem 1 HP Extension (nm) Stem 2 HP 10 nm Count Count Count The second step of U177 unfolding Isolated stem 1 hairpin unfolding 17.5 ± 1.0 pn 16.8 ± 1.4 pn 14.2 ± 1.2 pn Isolated stem 2 hairpin unfolding Unfolding force (pn) Unfolding force (pn) Stem 1 is disrupted during the second step of low force two-step unfolding
34 Single-molecule mechanical (un)folding pathway astem 1 hairpin b c Stem 1 hairpin Folding intermediate Native pseudoknot Native pseudoknot Hopping (10-20 pn)? Folding (3 pn) Folding (<10 pn) Single strand Unfolding (15-20 pn) Unfolding (15-20 pn) Unfolding (50 pn)
35 Analysis of one-step unfolding forces to map structural features of the folding intermediate state Low force two-step reaction (2 nd step) Unfolding force (pn) Delta U177 U Extension change (nm) High force one-step reaction Low force one-step reaction Are base triples and stem 2 formed at the folding intermediate state?
36 Base triples only affect mechanical stability of native pseudoknots C C C G U Count Count ± ± ± ± ± ± ± 1.9 CCCGU 33.9 ± 3.0 CCC GU U Unfolding force (pn) Unfolding force (pn)
37 Position of base triple matters ± C 38.1 ± C 102C Cou unt Count ± ± ± ± C U Unfolding force (pn) Unfolding force (pn)
38 At the intermediate state, base triples are NOT formed and stem 2 is partially formed a Stem 1 hairpin Folding b intermediate c Stem 1 hairpin Folding intermediate Native pseudoknot Native pseudoknot Hopping (10-20 pn) Folding (3 pn) Folding (<10 pn) Single strand Unfolding (15-20 pn) Unfolding (15-20 pn) Unfolding (50 pn)
39 Hopping of stem 1 hairpin and Folding intermediate of U177 observed at constant force Force (pn N) Extension (nm) nm H S Time (sec) Single Strand Stem 1 Hairpin Folding Intermediate
40 Hopping of stem 1 hairpin and Folding intermediate of U177 observed at constant force Force (pn N) Extension (nm) nm H (I) P Time (sec) Folding Intermediate Stem 1 Hairpin Native Pseudoknot
41 Similar mechanical stability observed for UAU, C + GC, and UGC base triples C175G 18.8 ± ± 3.1 U. G-C 114C175G 101C114C175G Count Count ± C114C175G ± ± ± 3.2 U Unfolding force (pn) Unfolding force (pn) C +. G-C U. A-U
42 Structures of major groove base triples U. A-U C +. G-C U. G-C
43 New folding intermediate structures appear for pseudoknots with mutations in stem C175G 18.8 ± ± 3.1 U. G-C 114C175G 101C114C175G Count Count ± C114C175G ± ± ± 3.2 U Unfolding force (pn) Unfolding force (pn) C +. G-C U. A-U
44 At the new intermediate state, base triples are NOT formed and stem 2 is fully formed 3 pn 3 pn Folding intermediate New folding intermediate Native pseudoknot
45 Multiple folding intermediate structures appear for TeloWT TeloWT U177 bulge Co ount Count ± ± ± 4.3 U Unfolding force (pn) Unfolding force (pn)
46 NMR reveals similar structures but different dynamics for TeloWT and U177 TeloWT U177 Kim, Zhang, Zhou, Theimer, Peterson, and Feigon, (2008) J. Mol. Biol.
47 Triplex structures significantly enhance mechanical stability of native pseudoknots. Triplex structures do not form in the folding intermediate states. Is there correlation between native pseudoknot mechanical stability and 1 ribosomal frameshifting efficiency?
48 Correlation between native pseudoknot mechanical stability and frameshifting efficiency 70-1 frames shift efficiency (%) CCCGU CCC GU U C114C175G 100C115C174G TeloWT (with U177) 102C 115C174G 101C 114C175G Average unfolding force (pn) y = (0.19 ± 0.27)exp((0.11 ± 0.03)x) R 2 = 0.84 x = 0 pn, y = 0.2 % x = 57 pn, y = 100 %
49 Unwinding of mrna pseudoknot by ribosome: Rotation is needed Frameshifting is enhanced by stabilizing both stem 1 and stem 2 3
50 Conclusions RNA pseudoknot unfolding and folding pathways were revealed by single-molecule and mutational studies. Both major groove and minor groove base triples of a pseudoknot contribute to mechanical stability and 1 frameshifting. Mechanical unfolding may mimic helicase activity of ribosome on mrna.
51 Acknowledgments University of California, Berkeley: Prof. Nacho Tinoco Prof. Carlos Bustamante Dr. Steve Smith Dr. Jin-Der Wen National Chung-Hsing University: Prof. Kung-Yao Chang Mr. Ming-Yuan Chou The Scripps Research Institute: Prof. David Millar Prof. James Williamson Prof. Larry Gerace University of Rochester: Prof. Douglas Turner Prof. Tom Krugh Prof. Scott Kennedy
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