1. Importance of a highly conserved
guanidine residue for thermo-switch
ability of RNA
Studied with fluorescence dynamics
in a mutant RNA thermometer
2. Riboswitch
-Regulators of gene expression
• In 5’ un-translated region of mRNA
• senses concentration levels of various small metabolites within cell
• control a large number of genes
3. Regulation types
• Activation or repression
• transcriptional using a
terminator hairpin
• or translational by
sequestering the Shine Dalgarno
region
ON
OFF
OFF
ON
ON
OFF
OFF
ON
4. RNA thermometer
• Ribo-switch regulating heat shock proteins
• At low temperature Shine- Dalgarno sequence
is sequestered by non canonical base pairing
• Undergoes 3D conformational changes at
elevated temperature that affect the
translational machinery.
5. 2-AP :the probe
Structure and photo physics:
• A fluorescent nucleobase
• can base-pair with uridine,
can replace normal A:U base
pair without substantial
deformation
• Provides the means to probe
structure and dynamics of RNA
Source: Kathleen B. Hall ,Methods in
Enzymology, vol469, 2009
6. 2-AP incorporated RNA
• complex photophysics
compared to free 2-AP:
1. Fluorescence is quenched
when 2-AP stacks with
another bases
2. Fluorescence decay becomes
multi-exponential when 2-AP
moves independently of
flanking bases
Source: Kathleen B. Hall ,Methods in Enzymology, vol469, 2009
7. Fluorescence intensity decay
• The fluorescence decay:
decay of excited state of
molecule to ground state is
given by-
I(t ) = I(0) exp(-t/τ), τ =1/(Γ+∑knr )
τ- information about the time
available for the fluorophore
to interact with or diffuse in its
environment
• measurements of 2 types:
1. Steady state
2. Time resolved
Source: principles of fluorescence spectroscopy, J.
Lakowicz
∑
8. Steady state vs Time resolved
• Steady state observation: an average of time dependent
phenomena over the intensity decay of the sample
• ISS =∫I(0)exp(-t/ τ)dt =I(0) τ
• Steady state measurements are simple, time resolved
measurements require complex instrumentation
• Why time-resolved measurements?
• ans: information lost during averaging process.
• Macromolecules exist in more than a single conformation, decay
time of bound probe depends on the conformation. The intensity
decay could reveal two decay times, and thus the presence of more
than one conformational state. The steady-state intensity will only
reveal an average intensity dependent on a weighted averaged of
the two decay times.
9. Time domain measurement
• width of the pulse is shorter than τ
• time dependent intensity is measured
following the pulse
• decay time τ is calculated from the slope
of a plot of log I(t) versus t
• polarizer orientation at 54.7⁰ from the
vertical z-axis to avoid the effects of
rotational diffusion and/or anisotropy on
the intensity decay
What so special about these measurements?
• Ans: most samples display more than one decay time
• suppose the probe residing in 2 slightly different conformer
shows 2 different decay times( 1ns & 5ns)
•The intensity decay is now a double exponential:
• goal of intensity measurements: recover τi & αi
Source: principles of fluorescence spectroscopy, J. Lakowicz
• In general :
10. 2-AP incorporated RNA
- The subject of the experiment
• point mutant RNA- Guanidine from position 15 deleted (mentioned as 15D)
• Adenine residue were replaced with 2-AP at positions 24, 27, 35; one at a time
• Recovered fluorescence observables: 1. mean fluorescence lifetime, τm(= Σαiτi), ∑i=1
2. anisotropy decay, r(t)
• Conditions: 1. At 20oC (with and without ribosome)
2. At 45oC (with and without ribosome)
3. At 450C, in presence of 7M urea
11. Site specific fluorescence kinetics of
free (unbound to ribosome) RNA
• Fluorescence intensity decay kinetics showed the presence of 4 lifetime components.
•
• persistence of structural integrity even at high temperature
• there is hardly any loss of base pairing at the thermometer region at high temperature
without ribosome.
12. Site-specific fluorescence analyses of
ribosome-bound RNA
• Binding to ribosome results in slowing down of the global tumbling dynamics of the RNA
• longer rotational correlation time (> 20ns)
• RNA binds to ribosome both at 20°C & 45°C :
13. Site-specific fluorescence analyses of
ribosome-bound RNA
• Mean life time , τm decreases at elevated temperature
• temperature dependent non-radiative processes become highly active at higher temperature
• RNA strand does not open up
14. Comparison of the mean lifetimes
• The much lower values of τm for the mutated RNA indicates much tighter base pairing
and more efficient base stacking
• So the highly conserved Guanidine residue introduces an inherent asymmetry within the
structure which is essential for thermo-switch action
15. conclusion
• The highly conserved guanidine residue
is really an essential for opening up of
the SD sequence
• it is the delicate synchronisation of both
the presence of that particular guanidine
residue & ribosome surface that makes
the initiation of translation possible.
