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Hot off the Press: Basepair resolution of RNAp transcription

by PhilipJ on 27 November 2005

In what is arguably the biggest news out of the optical trapping community in some time now, the Block lab at Stanford University has recently been able to resolve single basepair steps of the molecular motor RNA polymerase as it transcribes RNA from a DNA template!

As reported in this week’s Nature, a couple of key changes to their experimental apparatus have enabled the detection of 3.4 Angstrom steps, the distance between basepairs on a DNA molecule. The first of these changes are fairly pedestrian, they decoupled their experiment from their optical table by using two optical traps set up next to each other, as shown in this figure:

dumbells

By having two optical traps, they have greatly decreased the effects that drift will have on their signal. They are no longer directly coupled to the inverted microscope, which itself is sitting on a table, which itself is sitting on the floor, etc, etc. The other significant change in their instrument over standard optical tweezers is that the entire beampath is now filled with helium gas. The indices of refraction for regular air and helium don’t differ until their 4th decimal place, but this is evidently a significant enough decrease that beam pointing instabilities are greatly decreased, leading to significantly higher stability. In fact, when looking at the power spectrum of the noise in their system when comparing air to helium, their is nearly an order of magnitude difference for low frequency components (figure 1b in the article)! It also doesn’t hurt that the room they house their instrument is temperature controlled to within 0.1 degrees C!

stepsArmed with this significantly “quieter” instrument, the group looked at RNAp as it transcribed DNA to RNA. This time, instead of being unable to discerne a step size due to the large noisy position fluctuations, there are clear ~3.4 Angstrom changes in position, as a typical time trace to the left shows (dotted grey lines spaced 3.4 Angstroms apart are there to guide the eye). Looking at the spectral density contributions of position, they see a strong peak at 3.7 +/- 0.6 Angstroms, right around the distance between individual bases along the DNA helix!

With this significantly higher resolution measurement of RNAp dynamics, the paper then goes to try and address a specific model for transcription based on either a thermal ratchet or power stroke mechanism. Through Michaelis-Menten kinetics, it was clear that a power stroke (dashed green) mechanism was unable to model the observed dynamics, while an extended brownian ratchet (solid red) or regular brownian ratchet (dotted blue) model could explain the Forces and velocities observed.

As it has been described by others, this is a real tour de force paper. And best of all, a reprint is available from the Block lab’s website for free!



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