by PhilipJ on 27 March 2008
While we’ve been watching trippy 70s-era translation videos on YouTube, the Bustamante lab has been busy measuring this process at the single-molecule level, at least indirectly. This was one of those surefire Nature papers, whenever someone figured out how to measure translation at the single molecule level using optical tweezers, it would show up in Nature, and sure enough….
In Following translation by single ribosomes one codon at a time (closed access, PDF), we get to see for the first time the march of the ribosome along mRNA. I don’t think we’re anywhere near the end of single-molecule ribosome work, and part of the reason why is the setup for the experiments outlined in this paper. Reproducing part of Figure 1 from the paper:
What’s being measured is not the action of the ribosome itself, but the extension of the RNA/DNA construct as translation occurs. RNA/DNA handles are coupled to mRNA hairpins, and an initially stalled ribosome is then re-activated (by supplying the appropriate tRNAs), and the extension vs time of the entire construct is monitored at very high resolution. Examples of their extension vs time traces look like this:
The left hand plot shows a typical trace as the ribosome carries out its work, and the right hand side shows what’s called a pairwise difference distribution, or basically the difference between data points. If there is some periodicity in the dynamics, this would show up as a peak in the pairwise differences, and indeed they see peaks at 2.7nm (and multiples thereof). This corresponds to three basepairs being broken in the hairpin, and the ribosome translocating by 3 bases (where three bases form a codon, the unit which specifies for an amino acid). While not a surprising result (despite the authors claiming this was “striking”), this is still significant.
The tricky bit, however, is that this doesn’t really get at the heart of the ribosome’s interesting mechanochemistry. We still know nothing about the force-velocity relationship in single ribosome molecules, and while there are some interesting experiments in this paper involving dwell times at Shine-Dalgarno sequences, and some discussion on changing the ribosome’s reading frame, the paper is a little short on really new measurements.
Technically this is impressive, but the new and interesting data on ribosomes that single-molecule techniques can measure are still to come.
(Hat tip: in singulo)