PhD student in Molecular Evolution with Dr. Gemma Atkinson

Gemma Atkinson is looking for a PhD student in Molecular Evolution.

The candidate should have:

 - a Masters degree in a biological or computational discipline
 - a strong interest in, and enthusiasm for molecular evolution
 - familiarity with basic sequence and phylogenetic analyses
 - experience in using a programming language such as Python, Perl, Java etc
 - fluency in spoken and written English

The PhD will be funded by a monthly stipend, with additional monies available for regular attendance at international conferences and workshops, and for visiting labs abroad. Information on funding is available by request.

Applications should contain:

 - a full CV with detailed description of previous relevant experience
 - a statement of academic interests
 - an electronic version of the Masters thesis
 - the names and contact details of at least 2 referees

The candidate is expected to start at the latest September 2012. Please send applications and informal enquiries to gemma.atkinson@ut.ee

 More information about the research of Dr Atkinson can be found here.

Update: the call is closed, two applicants selected.

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Most of the mitochondrial proteins are imported from the cytoplasm, with only a small fraction (about 1%) encoded in the mitochondrial genome. Import is mediated by two complexes: TOM (transporter outer membrane) and TIM (transporter inner membrane). We have a pretty good idea about the players involved in mitochondrial protein import, but we have very little idea about the dynamics of TOM/TIM movement in the mitochondrial membrane.

We tried addressing this question using single molecule fluorescent microscopy in isolated yeast mitochondria. What we see is that Tom40, the central component of TOM complex, is highly confined (i.e. restricted in terms of aerea it can sample) but within its confinement it moves pretty rapidly.

References:

Kuzmenko et al., Scientific Reports (2011) 1:95

long memories of RelA

Enzymes have their cycles, catalytic ones: bind a substrate, catalyse some sort of reaction, release the product... then do it again. These cycles have memory effects: long turnover is likely to be followed by another long one, and short one is likely to be followed by another short one. This makes total sense: efficient act of catalysis is possible only when appropriate conformation is achieved and all the residues are aligned as they should be... and that is a recipe for one more efficient round!

Now let us look at RelA. Based on in vivo single molecule tracking investigations we recently proposed a model of RelA catalytic cycle: it sits on the ribosome, gets activated by arrival of deacylated tRNA to the A site, falls off and performs multiple acts of ppGpp synthesis from ATP and GDP. Importantly, RelA must go through the ribosome-bound stage in order to get activated. This seems to be an extreme cause of memory effects - while active off the ribosome, RelA remembers the activation event that happened on the ribosome!

It is quite a scary thought... what else does it remember? Does it remember the moment I started working on it? Well, surely not, even I don't remember that moment any more. Or may be I am just blocking out that memory.

References:

H. Peter Lu, Phys. Chem. Chem. Phys (2011), 13 pp. 6734-6749, PIMD 21409227

Brian P. English et al., PNAS (2011), 108(13) pp. E365-373, PIMD 21730169

Active role of the stringent response in antibiotic tolerance

From time to time we try killing bacteria with antibiotics. Most of the bugs die, but not all. These survivors fall into two categories: resistant bugs and tolerant bugs. Resistant bugs have specific mechanisms counteracting the drug: mutations in the target site, enzymes destroying the antibiotic, etc. Tolerant bugs are not getting killed using some more general approach, such as forming a biofilm efficiently shielding them from contact with the drug or shutting down its biosynthetic activity and waiting for the better days to come.

The stringent response is a mechanism rewiring the bacterial physiology under stress. It changes many things simultaneously, and, not surprisingly, functionality of the stringent response is linked to antibiotic tolerance. However, the big question here is the nature of this link: do bugs need functional stringent response in order to tolerate the drug just because relaxed bugs do not shut down their growth when needed and die, or does the stringent response induce production of certain specific enzymes protecting from the drug?

Recent report by Nguyen and colleagues seems to settle this question. Using series of in vivo experiments with E. coli knock-out strains deficient either in stringent response per se (knock-outs of RelA and SpoT) or in down-stream stringent response-regulated targets they show that the main source of antibiotic tolerance is not a general biosynthetic shut-down. Specifically, they identify two genes induced during the stringent response - superoxide dismutase (SOD) and catalase - to be crucial for bacterial survival in the presence of antibacterials.  What these do, they protect the bug from the hydroxyl radical. And build-up the latter was recently identified as a common mechanism causing the cell death during treatment by different unrelated antibacterials. 

References:

Nguyen at al., Science (2011) 334, pp. 982-986 PIMD 22096200

Kohanski et al. Cell (2007) 130, pp. 797-810 PIMD 17803904

DksA and ppGpp regulate transcription of both rRNA and r-proteins

During the stringent response ppGpp together with a small protein DksA bind to the RNA polymerase and down-regulate transcription of the rRNA genes. It makes sense - there is no need for more ribosomes if there are not enough amino acids. However, ribosomes consist not only of rRNA but also of ribosomal proteins, and if the cell stops making rRNA it would make sense to stop making the ribosomal proteins as well.

And it turnes out that ppGpp and DksA do that too. Makes total sense, again. It is quite rare that something about the stringent response makes total sense, but there you are.

References:

Lemke, J. J., Sanchez-Vazquez, P., Burgos, H. L., Hedberg, G., Ross, W., & Gourse, R. L. (2011). Direct regulation of Escherichia coli ribosomal protein promoters by the transcription factors ppGpp and DksA. Proceedings of the National Academy of Sciences of the United States of America, 108(14), 5712–5717.

The RelA/SpoT Homolog (RSH) Superfamily: Distribution and Functional Evolution of ppGpp Synthetases and Hydrolases across the Tree of Life

Stringent response is run by the RSH (RelA / SpoT Homologue) proteins, but there are more RSHs then just these two. Usually researchers were finding them using an ad hoc approach: take your favorite bug you worked with for 10 years, blast its genome with RelA gene, find anything that looks like RelA, test  it.

Finally there is a proper analysis of RSHs across the tree of life: The RelA/SpoT Homolog superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life by Atkinson GC, Tenson T, Hauryliuk V, PLoS ONE, 6(8): e23479. 

Here is the take home message:

• there are loads of different RSHs out there: we identified 30 subgroups! 
• all the RSHs out there are now are classified (for now, that is. New genomes are coming out every day, damn the progress!).
• Archaea, Bacteria, Eucaryotes: they all have RSHs. I repeat: Archaea too.
• there are the long RSHs (i.e. Rel, RelA and SpoT) and there are the short ones. 
• The short ones have either ppGpp synthesis or ppGpp hydrolysis domain. The long ones have both, but not always both are functional.
• by comparing the long ones vs the short ones we identified residues potentially involved in the inter-domain cross-talk in the long ones (the short ones have only one domain thus there is no cross talk there!).

The bottom line: if you work on a strange RSH protein from a strange bug, do check out our paper. 

Fig. 1.  Maximum likelihood phylogeny of the ppGpp hydrolase domain.  Subgroups are labeled and shading behind the branches shows the most common domain structure observed for those groups, as per the legend in the inset box. Symbols on branches indicate bootstrap support, as per the inset box.

Hurray to Gem! 

Observing protein synthesis inside the living mammalian cell

Biologists really love seeing things for themselves. Take for instance the central dogma: DNA - RNA - protein. It all well and good when represented as childish-looking blobs fooling around and passing amino acids one to each other, but how about it actually happening in the real 3D cell stuffed with other goodies? Well, obviously, people tried looking into the question.

The first option is you can to label fluorescently some components of the machinery - ribosomes, mRNAs, factors - and plonk the bug under the microscope. The problem here is that you can't really see what these components are doing - are they idle? are they active? are they so fucked up by adding the fluoresecnt label that now they are being rapidly degraded? One possible solution is to interfere with the cell inhibiting some crucial step the component of interest is involved in and see what happens to the distribution of the labeled component. For instance, one can add an antibiotic. The problem with this approach is that you a) interfere with the system b) interfere with the system c) antibiotics are often fluorescent, so you interfere with the system even more.

One solution that is often used in vitro for converting fluorescence signal into the distance signal is by using FRET, Förster resonance energy transfer (for an excellent review of FRET-based single molecule investigations of translation in vitro see Blanchard). The idea behind FRET is that one uses two fluorofores at the same time. Fluorofore 1 is excited at wavelength λ1, and emits at λ2. Fluorofore 2 is adsorbs at λ2 and emits at λ3. In order for all this cascade to work, the two fluorofores have to be very, very close because efficiency of FRET decreases with distance with an inverse 6th power law.

Now, back to observing translation in vivo. Barhoom at al. exploited the FRET strategy in their piece that just came out in NAR. They used a FRET pair consisting of two labeled tRNAs. These two are getting very close on the ribosome, generating a FRET signal (a strategy recently used in vitro by Uemura at al.). And they are getting close on the ribosome only if they are actively engaged in translation (Fig. 1). Therefore by looking at the tRNA FRET Barhoom and colleagues can observe spots of active translation inside the cell under different conditions, such as viral infection etc.

The paper is open access, so do take advantage of that.

Fig. 1. Two labeled tRNAs are constituting a FRET pair. When they are in close proximity on the ribosome they produce a FRET signal. When floating about in the cytoplasm, they are too far to generate FRET.


PS: this post is part of the MolBio carnival Nr 14!

References:

Blanchard SC (2009). Single-molecule observations of ribosome function. Current opinion in structural biology, 19 (1), 103-9 PMID: 19223173

Barhoom S, Kaur J, Cooperman BS, Smorodinsky NI, Smilansky Z, Ehrlich M, & Elroy-Stein O (2011). Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA. Nucleic acids research PMID: 21795382

Uemura S, Aitken CE, Korlach J, Flusberg BA, Turner SW, & Puglisi JD (2010). Real-time tRNA transit on single translating ribosomes at codon resolution. Nature, 464 (7291), 1012-7 PMID: 20393556