1. No category

RNA Folding and Action in
Real Time
Why thermodynamics is not
enough for understanding RNA
• Instructor
– Professor Pan Li
Office: Life Sciences 1108
Tel: 591-8879
email: [email protected]
Office hours: MW 1:30 pm-2:30 pm
or by appointment
Transcription Attenuation in E.coli trp
Operon
Yanofsky RNA 2007
OR
hairpin 1-2
Two tandem trp residues
in leader peptide
hairpin 1-2
hairpin 3-4
hairpin 2-3
Yanofsky RNA 2007
Why thermodynamics is not sufficient for
understanding RNA
• Alternative folding of RNA (many energy
minima)
• Heterogeneous distribution of energy and
physical states
• Non-equilibrium
– Need to understand kinetics
• How proteins/ligands/RNA act on RNA
Folding Energy Landscape of
RNA
Woodson, S.A. (2000)
Gralla & DeLisi (1974)
• Alternative folding is thermodynamically
inevitable;
• Rates of structural rearrangement vary and
depend on conditions (proteins, ligands etc)
– RNA can be kinetically trapped in misfolded
states
• Folding and structural rearrangement have to
compete with other processes, such as
transcription;
• Alternative folding and structural
rearrangement are biologically important.
RNA Structural Ensemble
• Distribution, partition function
• Thermodynamic or kinetic control
– Most stable fold dominates or fastest fold
dominates
• Protein, transcription etc
• In vivo or in vitro
• Kinetic control of riboswitches
– The speed of RNA transcription and metabolite
binding kinetics operate an FMN riboswitch.
(2005) Wickiser JK, Winkler WC, Breaker RR,
Crothers DM. Mol Cell. 18:49-60.
– The kinetics of ligand binding by an adeninesensing riboswitch. (2005) Wickiser JK, Cheah
MT, Breaker RR, Crothers DM. Biochemistry
44:13404-13414.
• In vivo vs in vitro
– Kinetics and thermodynamics make different
contributions to RNA folding in vitro and in yeast.
(2005) Mahen EM, Harger JW, Calderon EM,
Fedor MJ. Mol Cell. 19: 27-37
Difficulty to deal with heterogeneous distributions
• How to measure composition and
properties of a mixture? (lack of
measurable parameters)
• Which species in the mixture are
biologically functional?
• How to synchronize reaction/folding of a
mixture? (initiation, and process)
• Kinetics can be studied at bulk, especially
on well-folded structures such as
ribozymes.
• It is better to be don at single-molecule
level.
– Conceptually simple
– Survey a distribution instead of measuring
averages (repeat many times)
– measure distance as an indicator of
reaction/folding
– Manipulate structure of a single molecule
Why Single Molecule?
undocked
1 mole = 6.022 x
1023 molecules
<activity> of x mole molecule
Maxwell-Boltzmann Distribution
docked & active
Ensemble Kinetics
Tinoco et al. (2006) Quarterly Review of Biophysics
Single-molecule Kinetics
Tinoco et al. (2006) Quarterly Review of Biophysics
Single-molecule Techniques
• Mechanical unfolding by optical tweezers
• Single-molecule fluorescence microscopy
(especially FRET)
• Reference:
– Li and Tinoco (2008) Annu. Rev. Biochem.
77:77-100
– Joo et al. (2008) Annu. Rev. Biochem. 77:5176
RNA as a Small Rubber Band
Observables: f(t), X(t)
Pulling an RNA Hairpin
!G = reversible work
Lifetimes of a single molecule
at folded hairpin (short
extension) and single-stranded
states (longer extension)
Force Dependent (Un)Folding Kinetics
k(F) = k(0) e
F!x‡/kBT
dPreactant/dt = -kt
At Bulk:
d[Reactant]/dt = -kt
Li et al. Biophys. J. (2006)
At Bulk:
k(T) = k(0) e
!G‡/kBT
Physical Meaning of !X‡
k1
X
k-1
X
How many base pairs are
broken at the transition state?
Keq = [U] / [F]
0
F"X/kBT
= Keq e
0
F"X1‡/kBT
k1 = k1 e
k-1 =
0
k-1
F"X-1‡/kBT
e
Predicting Folding From Energy Landscape
Sequentially breaking base pairs
base open
loop open
kbaseclose
-!Go(basepair)/RT
= Keq (zip) = e
kbaseopen
kloopclose
-!Go(loop)/RT
= Keq (loop) = e
kloopopen
Assuming the distance to the transition
state for breaking a base pair is #0.1 nm
X‡close = X - X‡open ! "X
Matthews et al. J Mol Biol 288, 911
Bell Science 200, 618
Cocco et al. Eur Phys J E10, 153
Force Unfolding of Group I intron
Ribozyme
Onoa et al. (2003) Science 299: 1892-5
Nanomanipulation of Single RNA molecules
• Pull and relax like a rubber band;
• Quickly quench the force to induce folding
of less stable structures;
• Increase force again to refold;
• For large RNAs, use force to control
folding of individual domains, and tertiary
packing.
Fluorescence Resonance Energy
Transfer (FRET)
Dynamics of Hairpin Ribozyme
An single-molecule multiple turnover assay
Nahas et al. (2004) Nat. Struct. Mol. Biol. 11:1107:13
Sequential Actions of a Single Hairpin
Ribozyme
A single hairpin ribozyme molecule is washed with different
Mg2+ buffer and various [oligo]
Liu et al. (2007) PNAS 104: 12634-9
Effects of Other Molecules
• Important biologically (in vivo)
• Stabilizers
– Protein/ligands/RNA that recognize and
stabilize a structure domain, thereby
enhancing a particular fold
• RNA chaperones
– General helix destabilizing
– Allow RNA to attempt various conformations
CYT-19, a DEAD-box
Chaperone
Bhaskarah and Russell
(2007) Nature 449: 1014-8
CYT-19 Redistributes RNA
Folds
• CYT-19 unfolds both corrected and
misfolded structures, but misfolded is
unfolded faster. (Likely, the native fold
leads to tertiary packing and prevents
action by CYT-19)
• Under low [Mg2+], CYT-19 speeds up
misfolding, which is thermodynamically
less favorable than the native fold.
• Many other RNA chaperones. Some do
not use ATP.
• Helicases?
• Assembly of ribosome and other large
RNPs.