Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large

ribozyme

Jean chamoun

biophysical tools

April 20, 2004

Outline

• Single molecule FRET background

• Paper introduction

• Paper discussion

• Conclusion

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.

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).

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.

Streptavidin-biotin complex

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

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

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)

• 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.

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

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)

• 0.1--0.53, 0.4--0.59,1--0.60• but the peaks in the three

reveal the presence of interm.(0.3,and 0.7)

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.