1. ensemble distinctly guide RNA through preferred folding pathways
The electrostatic environment, native state topology and initial conformation
ensemble distinctly guide RNA through preferred folding pathways
Alain Laederach1, Inna Shcherbakova2, Magdalena Jonikas1, Russ B. Altman1, and Michael Brenowitz2
1Department of Genetics, Stanford University and 2Department of Biochemistry, Albert Einstein College of Medicine
Abstract:
While it is generally agreed that large RNA molecules typically follow multiple parallel pathways through rugged folding landscapes, the forces that partition molecules among these pathways are poorly understood. Direct influences on the physical forces that govern the folding reaction include native state topology, electrostatic environment and the ensemble of conformations present in the prefolded RNA. The relative contribution of these factors on folding flux partitioning was investigated for the Mg2+-mediated folding of the Tetrahymena ribozyme by coordinated time-resolved hydroxyl radical footprinting and kinetic modeling [A.L. Laederach, I. Shcherbakova, I., M.P. Liang, M. Brenowitz & R.B. Altman (2006) J. Mol. Biol. 358(4): ]. A single kinetic model topology with common intermediate structures is observed that is independent of the concentration or type of monovalent cation that is present during folding. The differences in the time-progress curves at the different ionic conditions are accommodated by repartitioning of the folding flux among the available folding pathways. Introduction of a severely destabilizing mutation changed the structure of the intermediates but not the kinetic model topology, demonstrating that the native state topology dictates the structures of the folding intermediates. The effect of initial RNA conformation and electrostatic environment upon folding on flux partitioning was distinguished from folding experiments in which the monovalent cation and Mg2+ concentrations were concurrently adjusted. The resultant kinetic models show that whereas native state topology determines the intermediate structures, their lifetime and relative abundance are dependent on either or both the initial conformational ensemble or the folding reaction conditions.
Introduction:
RNA size and shape are dependent
on the monovalent salt concentrations
RNA folding landscape has intermediates. These
can be identified through local measures of structure
Fast Fenton Footprinting measures
RNA Folding locally
The more surface
exposed nucleotides
will be more reactive
C144
G145
C146
A147
C148
C149
C150
G151
C152
U153
U154
G155
C156
A157
U158
C159
G160
A161
A162
C163
A164
U165
A166
G167
G168
U169
A170
Lot’s of smaller RNA
molecules
+ •OH
32P
Run on a Gel
SAFA is a great program
to analyze gels:
Conclusions:
Results:
To resove this paradigm, we perform
folding experiments where we start at one
Salt concentration (200 mM) and jump to 600 mM
Clustering of time-progress curves reveals
Differences in their shape
The partitioning of the local sites into
clusters is however conserved
The kinetic model predicts the time-evolution and
flux through the different intermediates
1.) Native StateTopology defines the structure
of the intermediates in solution
2.) The electrostatic environment (or counter-ion
concentration) adjusts the flux through the
different folding pathways.
3.) The initial conformational ensemble
controls the initial flux partitioning, but the
reaction conditions affect the rates
4.) This suggests an initial, condition dependent
partitioning of the molecules prior to folding.
And a single Kinetic Model topology
out of 84 fits the data using KinFold
Acknowledgements:
This work was supported by a program p
roject grant from the National Institutes of Health
(grant P01-GM-66275). This work was also partially s
upported through the NIH Roadmap for Medical
Research grant U54 GM Information
on the National Centers for Biomedical Computing
can be obtained from:
These results show that the flux through the
pathways is controlled by the initial conformation
while the rate is determined by the solution
conditions during the folding pathway.
These results suggest that while the native state
topology of the RNA determines the structural
nature of the intermediates in solution, the
electrostaticenvironment repartitions the flux
among these intermediates. However, we do
not know if this repartitioning is a result of the
initial conformation or of the conditions under
which the reaction is carried out.
Similar curves are obtained for L5b
mutant as well