Catalyzed and Uncatalyzed Mechanical Unfolding of RNA Molecules One at a Time

1
Catalyzed and Un-catalyzed Mechanical
Unfolding of RNA Molecules One at a Time
2
Mechanical Unfolding of Single RNA Molecules by
Force
Liphardt et al. Science, (2001) Onoa el al.
Science (2003)
3
Mechanical Unfolding of Single RNA Molecules

• RNAs must adopt specific 3-D structures to
  perform their
• structural and catalytic roles.

• RNA structure is hierarchical
• - Energies of 2ry and 3ry interactions are
  additive
• - Stability of 2ry interactions are independent
  of the
• tertiary context in which they are found.
• - It should be possible to predict its 3-D
  structure from its sequence
  

• Solving the RNA folding problem requires
  understanding
• the kinetics and thermodynamics of 2ry an d
  3ry elements.

4
T. thermophila Group I Intron
10
5
The P5abc Sub-domain
P5abc
p
p
p
p
p
p
p
p
p
P5ab
Mg
6
Mechanical System to Pull a Single Molecule
10 nm
Bead Radius 1000 nm
300 nm
A well-defined reaction coordinate end-to-end
distance RNA unfolding in the cell is largely a
mechanical process
7
Experimental Set-up
8
Reversible Pulling of P5ab
Handle behavior is well-predicted by
Force (pN)
Extension (nm)
9
P5ab Shows Bi-stability
15
Near or at the transition force, P5ab displays
bi-stability
Force (pN)
10
Bi-stable Length with Constant-Force Feedback
Directly observe k1 , k-1, and Keq
as a function of force under exp.
conditions
length
time
11
Modified vant Hoff Equation
Ratio of rate constants give the equilibrium
const. An expression equivalent to vant Hoffs
equation now holds
Where DL(F) is the difference in length between
the folded and the unfolded state.
DG -156 8 kJ/mol
DL 23 4 nm
(in Mg)
MFOLD -147 kJ/mol
12
Effect of Tension on the Rate of Unfolding
An equivalent expression to Arrhenius equation
can be derived
Where Dl1,-1? is the distance from the folded
to the transition state
k1
Closed
Open
k-1
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Effect of Tension on the Refolding Rate
k1
Closed
Open
k-1
The transition state is equidistant
between the folded and the unfolded state
Dl1? (F1/2) 11.2 nm
Dl-1 ? (F1/2) 10.9 nm
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The systems
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P5abc
p
p
p
p
p
p
p
p
p
P5ab
Mg
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Mechanical unfolding of P5abc
In (EDTA)
with Mg
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Pulling at Different Rates Gives Distance to
Transition State
Loading rate (r) Stiffness of trap
X rate of pulling
where,
Dl 1.6 nm
(folded --gt )
Tertiary interactions make structures
brittle, i.e., require higher forces to break,
but smaller deformations.
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Unfolding Trajectory of P5abc
(intermediate)
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P4-P6 complete folding domain
19
25
Multiple Kinetic Barriers
Frequent paths
Less frequent paths Kinetic intermediates
Structure assignment?
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Unfolding the P4-P6 domain
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Assigning the Transitions
Use mutants or oligonucleotides
to eliminate tertiary or secondary contacts
22
Reconstructing the Unfolding Patwhays of the
Full Ribozyme
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Unfolding the P4-P6P3-P8 P9 Domains
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30
Unfolding Map for P4-P6P3-P8 P9
Arrows thickness give a measure of the prob. of
a particular path
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Direct Observation of Substeps Reveals the RNA
Unwinding Mechanism of HCV NS3 Helicase
Sophie Dumont Wei Cheng Ignacio Tinoco
Jr. Carlos Bustamante
HHMI

• Victor Serebrov (Yale)
• Rudy Beran (Yale)
• Anna Marie Pyle (Yale)

Dumont et al. Nature (2005)
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Helicases as Motor Proteins

• NTPases play central roles in a number of
  cellular
• processes. Molecular motors, generating
  mechanical
• work to translocate on nucleic acids and
  unwind them.
• Ubiquitous (1 of our genome)
• DNA metabolism replication, recombination,
  repair
• RNA metabolism pre-mRNA splicing, translation
  
• initiation, viral replication, etc.

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NS3 Helicase from Hepatitis C Virus

• N-term protease domain (181 aa) and a C-term
• helicase (456 aa), genotype 1a.
• Essential for viral replication in vivo of
  Hepatitis
• C virus drug target
• Belongs to the Superfamily II and the DExH
• family of helicases. 3' to 5' RNA DNA
  helicase
• Monomer one NTPase covers 8nt RNA
• Binds RNA as monomer
• Oligomeric form unknown in vivo

dU8
Serebrov Pyle, Nature 2004 Levin Patel, J.
Biol. Chem. 1999
Kim et al., Structure 1998
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Main Goal

• Study the single molecule (in singulo)
  dynamics of NS3 and address
  translocation mechanism

Specific Questions

• How does NS3 translocate on RNA?
• 2. How does ATP binding coordinate both
• translocation and unwinding?
• 3. How does unwinding occur within a cycle, i.e.
  at once or in small increments?
• 4. How does NS3 unwind passively? Actively?

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Our Strategy
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• Apply constant force to the ends of
  an RNA hairpin substrate with optical
  tweezers and measure end-to-end extension as
  NS3 unwinds the substrate in the presence of ATP.

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Experimental Setup
Resolution 20 ms, 2 nm (2 bp at 17 pN)
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Experimental Setup (detail)
5
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The Experimental Cycle
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Force unfolding
Helicase-catalyzed unfolding NS3 20 nM ATP
1 mM
Helicase buffer 0.9 glycerol, 0.1 Tween20, 30
mM NaCl, 0.75 mM MgCl2, 20 mM MOPS pH 6.5, 2 mM
DTT, 22C
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Necessary Controls

• Verify that any changes in extension of the
  substrate
• are due to unwinding of the RNA hairpin by NS3

?Extension
No hairpin, no activity 1bp hairpin 1nm (at
17pN)
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NS3 Unwinding in Real Time
50
40
30
Extension (nm)
20
10
0
0 2 4 6 8
10 12
Time (s)
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NS3 Walks in 11 bp Steps
35
Worm-like chain end-to-end distance in nm to bp
Pairwise-distribution analysis
Fourier transform yields step size
Step size 113 bp, average over gt100 traces
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NS3 Dissociation and Slips
Dissociation
Slipping
Pushed back?
Finite processivity
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Backward Movement?
Backward movement without reaching the end
Backward movement after reaching the end

• What is the origin of the backward movement?
• NS3 turns around the tetraloop?
• NS3 steps backward (now 5 to 3')?
• NS3 switches strand?
• NS3 dissociates in sequence?

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Are 11bp Steps the Miminal Stepping Units?

• Does the step size depend on
• Sequence? Design other substrate sequences
  with different mechanical unfolding patterns
• Force? Vary force
• ATP? Vary ATP, below/above KM 0.1 mM
• NS3? Vary NS3, below/above Kmonomer,RNA 6
  
• nM and Kdimer 19 nM (NS3/RNA
  NS3)

39
Designing New Substrates

• Design and make RNA substrates with barriers
• RNA1 no barrier, unfolds at once one rip
• RNA2 one barrier two rips
• RNA3 two barriers three rips

RNA2
RNA1
RNA3
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Walking on Different Substrates
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Step size 113 bp 34 traces
Step size 123 bp 38 traces
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Step Size Robustness
Step size independent of ATP
Step size independent of force
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Effect of NS3 Concentration
Stable entity performs unwinding
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Effect of ATP on Pause Duration and Step
Velocity
step
ATP affects pauses and steps
pause
44
Pause Duration and Stepping Velocity Depend on
ATP
14 4 bp/s
Mpd 3.9 s
20 8 bp/ s
Mpd 1.7 s
Mpd 0.6 s
44 17 bp/s
ATP binding during step?
ATP binding during pause
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What Occurs During a Pause?
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ATP binding
conformational change
same as bulk value
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Do Additional ATPs Bind During the Stepping?
Are there any discrete events within an 11 bp
step?

• Lower the concentration of ATP even further
• - Improve the resolution of the measurement

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Substeps And subpauses Revealed at Low ATP
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Subpauses Substeps at Low ATP
0.05mM ATP 35 traces
0.425 0.003 s
explains ATP binding during step evidence
suggesting 1ATP per substep
Substep 3.61.3 bp, 3 substeps per step
consistent with DGATP ltlt 11x DGbp
suggests a poss. helicase mechanism!
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Processivity Depends Strongly on Force
Lower back-pressure of hairpin on NS3 at higher
force increases processivity? NS3 is a
processive ssRNA translocase!
50
Force Affects Neither Pause Duration nor Stepping
Velocity
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Strand separation is not rate limiting
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RNA Substrates to Probe Sequence Effect
RNA-AG
RNA-GA
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Probability of NS3 Dissociationalong Duplex RNA
RNA-AG 30 A?U-30 G?C
5 nM NS3, 1 mM ATP 7 pN, 96 traces
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Substrates to Probe Barrier Sensing
RNA-6CG
RNA-3CG
RNA-6CG
RNA-3CG
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RNA-6CG -3CG Unwinding
RNA-6CG
RNA-6CG
RNA-3CG
RNA-3CG
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Probability of Falling Off Depends on Barriers
ahead of the Helicase
RNA-AG
RNA-3CG
7pN 1 mM ATP
RNA-6CG
RNA-6CG -3CG identical sequence up to this
point, but 40 drop already in fraction of
unwinding
RNA5 96 traces RNA7 58 traces RNA8 106 traces
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30GC (strong), then 30AU (weak)
Force 17pN
30AU
30GC
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Model Inchworm-Like
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Active NS3 complex has two RNA binding
sites translocator site and helix opener
site. or reverse orientation?
2-5 bp substeps
11 bp steps
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Model Inchworm-Like
NS3 monomer has two binding sites translocator
(11bp steps) helix opener (2-5bp substeps)
PAUSE dsRNA contacts NS3 every
11bp ko(1.90.1)s-1, kb(ATP)(9.91.0)103M-1s-1
SUBSTEPS NS3 walks on ssRNA 2-5bp at a
time STEP Vmax (436)bp/s, KM(ATP)(12050)µM
PcrA
NS3h
Kim et al., Structure 1998
Velankar et al., Cell 1999
59
Conclusions

• NS3 monomer is a processive single-stranded RNA
  translocase.

• NS3 translocates with a step size of 113 bp.

• Pause exit (translocator) requires two events
• ATP binding and an ATP-independent step.

• Stepping velocity (helix opener) depends on
  ATP
• via substeps of 2-5 bp which each require 1
  ATP.

• NS3 is a weak helicase it must receive help in
  the cell!