Wednesday, August 10, 2011

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

Thursday, August 4, 2011

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!


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