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
