1. Jean chamoun biophysical tools April 20, 2004
Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme
Jean chamoun
biophysical tools
April 20, 2004

2. Outline Single molecule FRET background Paper introduction
Paper discussion
Conclusion

3. Fluorescence resonance energy transfer (FRET) A powerful tool for measuring distances in the range 1-8 nm. Two fluorophores interact via induced dipole-dipole interaction.

6. Single-Molecule FRET Breakthroughs
1996, First successful single-molecule FRET experiment (Ha et al).
1999, First link between single-molecule FRET and distance (Deniz et al).
1999, First observation of structural changes via single-molecule FRET (Ha et al).

7. Why study biological processes at the single-molecule level?
Ensemble measurements average properties.
Single-molecule methods can be used to study
Detection of multiple kinetic paths
Transient intermediate states
Conformational changes
In general, they can lead to quantitative understanding of complex biological phenomena.

8. Streptavidin-biotin complex

9. Catalytic domain P RNA
Large enough to exhibit folding phenomenology of large RNAs
previously shown that there is three state that govern this folding U, I, and N
More knowledge of the intermediate states is crucial to understand the folding of large RNA
in other words the objective of this study is to elucidate the U-to-I transition in the equilibruim folding of P RNA

11. Ensemble measurement
First to establish if the FRET labeling interferes with the Ribozyme folding
Second to verify the Mg2+ concentration range associated with early stages of folding
And finally to provide results to compare with the single molecule experiments

12. Results(1)
This picture verifies the FRET is sensitive to Mg2+ concentration as seen in previous papers
Mg2+= 0mM(U) (FL emission dec.)
Mg2+ = 10 mM( F) (increase in Cy3 emission)

13. The manipulation of the RNA ( 3’ extension and its complementary DNA) shows almost no interference with the catalytic activity of the P RNA, the Mg2+ midpoint where 50% of the ribozyme are active is 1.5mM compared to 1.3mM with no alteration.
I-to-N transition occurs at [Mg2+]= 1.5mM meaning that the U-to-I transition occurs below this con.

14. Single molecule trajectories and FRET histograms(1)
Five Mg 2+ con.
Green is the donor and red is the acceptor
E(fret) trajectories are identified as Ia/(Ia+Id)
The difference in the single molecule trajectories(major conformational changes among intermediate, minor con.fluctuation within specific state

15. Single molecule trajectories and FRET histograms(2)
At the 0 and the 5mM con. We a narrowly distributed histog. That peaks at 0.1 and 0.85 which define the FRET signature for U and N( distance R)
, ,
but the peaks in the three reveal the presence of interm.(0.3,and 0.7)

16. Conclusion
They were able to show that there is more than on intermediate in the U-to-I state.
Complex inter and intra-class dynamics were observed for the equilibrium conformational fluctuations of the C-domain of P RNA.