Optical Tweezers: gene regulation studied one molecule at a time

Speaker: Steven Block
Department: Departments of Biology and Applied Physics, Stanford University
Subject: Optical Tweezers: gene regulation studied one molecule at a time
Location:
Delft University of Technology
Date: 1 December 2016
Author: Romano van Genderen

As the main program of the Kavli colloquium, we had guest lecturer Steven Block flown in from the US. The talk started with some quick introductions about him personally, including the fact that he is a member of JASON and advices the US government on science, a job he’s absolutely going to loathe next year. Afterwards, he immediately pulled us in with a simple analogy. Say you have a ship you want to sail from New York to San Fransisco. A lighter ship can go through the Panama canal, while a larger ship has to sail around South America to get there. But if you average the trajectories of all ships sailing between these two cities, you get a weird track going right through the Amazon rainforest and up the Andes. He concluded: Ensemble measures can be very misleading.

Next he introduced his instrument of choice, the optical tweezers, a device that allows very small movements and forces by steering a bead using a laser. But it can also be used to measure very small forces and displacements. Where do you find these displacements in nature, you ask. Professor Block said that these small steps are the key to the Central Dogma of biology, where DNA gets transcribed into RNA and translated into protein. All proteins acting in this process are motors, and the one he is focused on right now is RNA polymerase, for it plays a large part in gene control. The assay he uses for his optical tweezers is called the dumbbell assay, two beads with exactly one molecule of DNA in between.

One of his most influential researches was called the Great Step Hunt, or so he likes to call it. The question was, does RNApol make single nucleotide steps when reading DNA, or does it make jumps? By cooling down the experiment using helium and using a passive force clamp to make the spring constant of the trap exactly zero, he discovered that the RNApol makes steps of about 4.3 Å, so steps of 1 BP.

Afterwards he studied the folding of RNA into hairpin structures. Using the optical tweezers to pull open the hairpins, he found out that RNA folding can be effectively modeled as a thermally driven 2 state system, where P_open and P_closed are dependent on temperature, force and ratio of GC to AU bonds in the RNA.

His final subject was riboswitches, or pieces of RNA that regulate gene transcription based on the presence of adenine. If adenine is present, it binds a specific part of the RNA, the loop of a specific hairpin, which stabilizes the structure. But when it is absent, the chain is less stable, so it forms a hairpin further downstream, which isn’t formed when adenine is present. This hairpin interacts with the RNApol and shuts it down.

https://nanobiologyhonoursprogrammeblog.files.wordpress.com/2016/12/89f6c-iu.jpg?w=648

Figure 1.

A. The two configurations of the adenine riboswitch, either with bound adenine or without bound adenine and a terminator hairpin

B. The setup used by Frieda and Block to measure the RNA length

C. The two results of the riboswitch, termination without adenine and run-through with adenine

Source: K. Frieda and S. Block, “Direct Observation of Cotranscriptional Folding in an Adenine Riboswitch”, Science, vol. 338, no. 6105, pp. 397-400, 2012.

Using theoretical physics, mostly the Jarzynski theorem, he found that one specific GC-pair is the keystone that holds the entire structure together. But he also acquired data by measuring the length of the RNA leaving the RNApol. Those two pieces of information were combined to form the model of riboswitch behavior previously mentioned.

Currently he is studying another kind of riboswitch, namely the TPP riboswitch. But to study this one, he used another technology. By using FRET, he discovered the way the arms formed by the RNA hairpinning move and eventually bind each other tightly. This shows that there are many different riboswitches and there is still a lot to learn.

I really enjoyed this presentation, not only because professor Block is a very engaged speaker who really grabs your attention, but also because this topic is what nanobiology is basically about, integrating physics and biology. No applications or something of that order were given, but I didn’t care. For the first time in a long time I was totally engaged by his stories and theories, not even asking myself the question I often ask, namely “What can you do with it?”. In conclusion, it was a very amazing talk.

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