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.

New Principles of transcription coupled DNA repair

Speaker:  Evgeny Nudler
Speaker Institute: Howard Hughes Medical Institute
Department: Bionanoscience Deparment
Subject: New Principles of transcription coupled DNA repair
Location: Building 58 (TU Delft)
Date: 24-11-2016

Author: Nemo Andrea  

Evgeny Nudler is a researcher working at the Howard Hughes Medical Institute. He has a PhD in biochemistry and does active research in the areas of Molecular Biology and Biochemistry. He gave a talk on Transcription Coupled Repair (TCR), a process by which the transcription of genes facilitates detection and removal of DNA damage.

The first part of the seminar covered the current and past understanding of Nucleotide Excision Repair (NER) and the factors that recruit the NER system towards sites of DNA damage. This made the lecture quite accessible, as this meant everyone had a good overview of the current understanding of these mechanisms in vivo. This brief section was, for me personally, quite enlightening. We had covered NER in various courses but this short recap greatly expanded on the interactions of NER and RNAP and various other factors. It was also fascinating to see how various components we had studied in various courses such as magnesium atoms in proteins are critical in the functioning of various mechanisms.

The main focus of his research was how transcription and the corresponding movement and stalling at sites of DNA damage of RNAP results in the recruitment of the NER complex. He introduced various systems related to RNAP and its stalling that were not covered in the nanobiology courses such as GreA and GreB. He touched upon the well known concept of how the stalling of RNAP results in the recruitment of factors and subsequent alleviation of DNA damage, but also stressed how other elements moving along the DNA such as the replisome, can cause double stranded breaks when they come into contact with a stalled RNAP complex. He stressed how double stranded breaks have strong detrimental effects on the cell and that therefore stalled RNAP complexes also had to have a way to transition from a stalled to active state again. The old model, which had been proposed in 1993 by Selby and Sancar (science, 1993), was centered around a protein called Mfd and featured a system in which the RNAP is pushed over the site of DNA damage, where after the NER complex would be recruited. In other words, this model was based on forward dislocation of the stalled RNAP.

The new model which Nudler and his lab proposes differs significantly from the old model, but still features RNAP stalling at sites of DNA damage as the event that recruits the NER complex. This new model is centrally based off the two proteins NusA and UvrD. These proteins would push the RNAP backwards rather than over the site of DNA damage, exposing the site in this backwards dislocation manner. He presented evidence for this in various experiments that showed that if UvrD was removed, cells became significantly more sensitive to DNA damage. Additionally, if factors such as GreA/GreB and Mfd were increased in cells, the cells also became more sensitive to DNA damage. This is because, as he explained, these factors are counter-backtracking factors – they prevent the RNAP from dislocating backwards, a fact that has been confirmed by other studies.

They then set out to figure out exactly how these factors could facilitate the backtracking of RNAP. To answer this question, they employed what Nudler called a ‘power technology’ that is known as XLMS to figure out where the proteins such as UvrD could bind to RNAP. Analysis by this method resulted in a probable binding site for UvrD on the ‘backside’ of the RNAP molecule (on the side of the RNAP molecule furthest away from the direction of movement). They also determined that a single UvrD molecule is not good enough to explain the backtracking of RNAP, suggesting that dimerisation may be required for this model to reflect the system. They found out that in dimer form, this complex consisting of two UvrD molecules and NusA could indeed facilitate the backtracking of RNAP, but also determined that this dimer form is unlikely to persist for a long time due the the low concentration of UvrD in the cell.

They also realised that this system must be very flexible, as the activity of TCR various greatly depending on the conditions of the cell (e.g. in case of cytotoxic stress, the system is very active) so they set out to find some of the regulating factors. They found out that the bacterial alarmone ppGpp played an important role in regulating this repair system. Given ppGpp’s function as an alarmone, this would certainly seem feasible. After that, they uncovered the system by which the ribosome trailing the RNAP is removed from the complex. This removal is critical, as this ribosome is closely connected to the RNAP and could prevent the backtracking functionality. Lastly, they uncovered mechanisms by which the stalled complex could be reactivated, preventing DNA damage by the replisome colliding with the stalled complex. They found that by hydrolysis of ppGpp and by introduction of counter backtracking factors such as Mfd and GreA and GreB could restart the stalled RNAP

[1] Left: Experimental Data and the new model; [2] Right: on the effect of ppGpp

It was very interesting to see various theoretical concepts be used to decipher important mechanisms inside the cell. Powerful concepts such as dimerisation, catalytic sites and NER all coming together to make a new model that more accurately reflects reality. It was also interesting to see how new technologies such as XLMS greatly facilitate new discoveries and open up new possibilities.

If you wish to read his full article on (part of) this topic, visit:
[1]https://www.ncbi.nlm.nih.gov/pubmed/24402227
[2]https://www.ncbi.nlm.nih.gov/pubmed/27199428