Modeling of the ribosome and the RNA polymerase molecular motors

Speaker: Bahareh Shakiba
Department: Institute for Advanced Studies in Basic Sciences, Zanjan, Iran
Location: TU Delft
Date: July 12, 2017
Author: Teun Huijben

Bahareh has just finished her PhD and is now doing a Postdoc at the IASBS in Iran. In this talk she explained what she has studied during her PhD, in which she was interested in the two most important molecular motors present in the Central Dogma: the RNA polymerase and the ribosome. Both proteins can be seen as molecular motors since they use energy to move along a polymer (either DNA or mRNA). She was especially interested in the dynamics of these motors, so their pausing-behaviour and force-dependence.

The first part of the talk was about modeling the RNA polymerase (RNAP). Bahareh proposed a model to simulate the transcription steps using two components of the RNAP: the bridge helix and the trigger loop, which are both located very close the active site of RNAP (see Figure 1). The bridge helix promotes opening of the double stranded DNA helix and the trigger loop binds to the DNA and drags the DNA through the polymerase. The internal movement of components in the active site of RNAP during the transcription of a single nucleotide can be divided into multiple steps, which each having a energy barrier. Using the energy barriers calculated by other people, she computed the average duration of a pause during transcription. The average length of these pauses was the same as found in experiments.

 

ÈFigure 1: The bridge helix and the trigger loop are located near the active site of RNA polymerase. [M. Thomm, University of Regensburg]

However, I have some critical notes on this finding. First of all, she didn’t explain the model very well, so it was unclear to us in what way this model was new. Especially given the fact that many people do research on this subject and multiple models already exist. Besides that she didn’t mention the reaction rates she used in the simulations and where they came from.

Another important aspect is that she didn’t show the distribution of pauses. Normally it is very interesting to look at a histogram of pauses, to see how they are distributed, this can be Gaussian, exponential or Gamma, for example. Seeing the distribution will give a lot of information about the underlying processes and indicates if it is fair to simple take the average pausing time and compare this with experiments.

The second part of her presentation Bahareh talked about modeling ribosome dynamics. A ribosome is a protein complex that translated the mRNA to a protein. Bahareh studied how  mRNA-hairpins influence the processivity of the ribosome, since hairpins are formed in single stranded RNA and form roadblocks in front of the ribosome.

The active site of a ribosome has three free places to bind a tRNA molecule: the E- (exit), P- (polymer) and A-(active)-site (see Figure 2). The ribosome moves forward by first displacing the large subunit, followed by movement of the small subunit. If the mRNA in front of the ribosome has internally formed a hairpin, this can either result in necessary unwinding of the hairpin or a frameshift of the ribosome. The latter means that the ribosome temporarily detaches from the mRNA, resulting in errors in the produced protein.

hairpin

Figure 2: The active site of the ribosome has three active sites, the E-, P- and A-site. A tRNA loaded with an amino acid can bind to the ribosome and transfer its amino acid to the growing peptide chain. A hairpin in front of the ribosome. [Shakiba 2016, arXiv: 1607.0719v1]

There are currently two ideas of how ribosomes solve the hairpins. One states that the moving ribosome applies a force on the hairpin forcing it to unwind. The other theory states that the ribosome itself actively uses helicase activity to make the mRNA single stranded. Both theories are simulated in the model. Then she compared the model with experimental data of ribosomes translating mRNAs having hairpins, while applying a force on the mRNA. This comparison revealed that the model with ribosomes having helicase activity did the best job in explaining the experimental data. Giving the indication that ribosomes indeed have their own helicase activity.

As already stated in this report, the talk of Bahareh was quite hard to follow, especially because she didn’t explain the models very well. Therefore, the first part of her talk didn’t really impress me, since I didn’t see what was new and surprising in her model. However, the second part was easier to understand and got a clear, well described message.

Optical Tweezers: gene regulation, studied one molecule at a time

Speaker:  Steven M. Block
Institute Speaker: Applied Physics and Biological Sciences, Stanford University
Organising Department: Kavli Institute
Subject:  Optical Tweezers: gene regulation, studied one molecule at a time    
Location: Faculty of Industrial Design, TU Delft
Date: 01-12-16

Author: Nemo Andrea

Professor Block is a well-known figure in the bionanoscience department. He is considered to be one of the founders of the single molecule biophysics field. He currently holds the Ascherman Chair in the Departments of Applied Physics and Biology at Stanford University. One of his former students is Elio Abbondanzieri, who is currently one of the members of the bionanoscience department at the TU Delft. He gave an intriguing seminar on Optical tweezers and their applications and discussed riboswitches.

In the introductory section, Professor Block discussed the importance of the single molecule biophysics field, and how bulk analysis can have it shortcomings. This was followed by a short section on one of the workhorses of this field: the optical trap. Having been introduced to the working concept and applications of these optical traps in our course, it was interesting to see someone who pioneered the use of optical tweezers.

The more contemporary discussion started with the versatility of RNA polymerase (RNAP), which coincidentally was also the focus of the preceding week’s BN seminar. This RNAP molecule, as he explained is a very well document and very versatile protein complex which is regulated in part by RNA hairpins. To demonstrate both the applications of optical tweezers and the properties of DNA he explained how they could determine how many DNA molecules were tethered in their optical trap by measuring the force-distance curves and comparing it to the mechanical properties of DNA. This is not a new experiment by any stretch of the imagination, but it is key in illustrating the use of the optical trap.

The second part of his seminar revolved around the research done in part by Elio, namely researching the manner in which RNAP moves along the DNA, in particular focusing on testing the validity of the inchworm model. He explained the particular circumstances that made that experiment particularly challenging. To hear about the complexity of environmental noise and finding new ways to minimise noise in experiments was refreshing and educational, as we often solely see elegant and low noise data but never hear about what problem solving steps were taken in the actual experiment that ultimately lead to said results.

The Final and main focus of his seminar revolved around RNA hairpins and riboswitches. He explained how various theoretical formulas can be used to determine many properties of various RNA hairpins. They studied riboswitches, with a particular focus on a riboswitch that as adenine as its substrate. The idea behind this particular type of riboswitch is that when the substrate binds to the RNA molecule, it will affect the transcription of the gene itself. This way we have a way in which RNA molecules can become part of the complex gene regulation networks. This concepts also ties in nicely with the RNA world hypothesis.

While this seems feasible in theory, it is not enough to just describe a model qualitatively in order for it to be accepted. It must be mentioned that this is still an area of active research and only three riboswitches have currently been studied in any detail. Steven Block and his lab and others set out to characterise the mechanical properties and kinetic behaviour of the adenine riboswitch, with the goal of being able to describe it more quantitatively.

They studied force extension curves of the specific riboswitch (which ultimately is just RNA being transcribed) by using optical tweezers. They used a framework (Dudko 2006) [3] to find the free energy of activation and characterise various other aspects of the conformations of this RNA molecule as it would be transcribed. This ultimately lead to the creation of a 5 state system as described in a paper by Greenleaf et al. (2008). This group created an energy landscape of this system which elegantly describes the behaviour of this system and the effect of adenine on the energy levels.

Left: the adenine riboswitch as depicted in [1], right: the TPP riboswitch as seen in [2]

They also ran experiments on a different riboswitch, one that is found in eukaryotes and functions with TPP as a substrate. Structural analysis of this molecule had led to the proposal of a model wherein two hairpins would (blue and red in the image above) would come together in order for the riboswitch to be switched on. In order to verify this hypothesis, they combined the optical trap with FRET. They labeled each arm and observed the intensity and wavelengths of light emitted. This allowed them to make a model which suggests that there are various conformations in which this riboswitch can reside. More can be read about this in reference [2].

It was intriguing to see the results and applications of optical tweezers, a topic we have covered in physics courses and are heralded as one of the prime example of the nanobiology field and the new technologies that are created when the worlds of molecular biology and physics meet. Additionally, I had never explored riboswitches in any great detail, making this topic a completely new area for me. While they may only be a uncommon way to regulate gene expression, the mechanisms through which they regulate the genome are very unique and certainly require further study. Even with the things above, the thing I enjoyed most was seeing how theory led to experiments which then led to new theories and models.

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692365/
[2]https://elifesciences.org/content/4/e12362
[3]http://journals.aps.org/prl/abstract/10.1103/PhysRevLett.96.108101