Biocurious

/ a biophysics blog

Hot off the Press: Single-ribosome translation

Posted 26 March 2008 by PhilipJ under &

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:

single ribosome experiments setup

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:

ribosome steps

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)

  

  1. Anonymous    30 March 08    #

    From above: “the paper is a little short on really new measurements”

    You seemed to have missed the figure with the translocation time distribution… this could only be observed using high-resolution single-molecule techniques.

    This seems to me to be the most important and novel data in the paper.


  2. PhilipJ    30 March 08    #

    Hi Anonymous,

    It wasn’t missed, it just isn’t a critical result to the paper, as evidenced by the pure speculation offered up in their analysis: “The kinetic processes we identified here could involve, for example, steps in the unlocking rearrangement of the ribosome; or it is also possible that the ribosome moves over the length of one codon with three consecutive, equally timed, single-base substeps.”

    It will be interesting when these kinds of processes are distinguished, but this paper felt (to me, you may feel differently) that this was a “we finally got this to work!” paper, more than a “here’s some biologically relevant new data on how ribosome’s work” paper. That’s the nature of these kinds of experiments, so I don’t mean to belittle the result—I’m sure it has been a long, technical challenge to get anywhere with this system. I just feel like the best is yet to come.


  3. spooner    9 April 08    #

    Single-base substeps were supposedly seen by examining the histogram of translocation time (Figure 2d) and fitting for 1,2, and 3 sequential exponential processes.

    Data were taken at 200 Hz (5 ms between points) and each bin is 25 ms wide. That means the decision to categorize an event as a translocation with time less than 25 ms was based on only 5 points. Indeed, the events in every adjacent bin differ by, at most, only 3 points. This categorization must be done with a nontrivial amount of noise in each trace.

    My question: how does one justify this analysis? Does no one else take issue with this? Why isn’t the robustness/accuracy of their binning algorithm tested by comparing against simulated data to prove to me this isn’t a measurement artifact??

    I was under the impression that histograms of this sort (i.e. single molecule helicase motion along DNA/RNA) tend to undercount the lowest bins because of noise and step finding uncertainties…


  4. JohnJ    10 April 08    #

    Spooner, after re-reading the paper, they never claimed to see single-base substeps. Where exactly did you read that?


  5. spooner    10 April 08    #

    Apologies, I didn’t mean to say single substeps were directly observed on the traces themselves. Rather, because the translocation time distribution is best fit by a triple exponential process, the paper states this indicates three identical substeps of k=40/s happen in each codon step. “…no technique has allowed characterization of the dynamics of ribosome stepping to this degree of detail.” (page 4) This k gives a mean lifetime of 0.025 s for each substep (the bin width), which also adds to my suspicion…

    As was pointed out before, this is an awesome experiment and is in Nature because its totally cool and hard to do, but one could argue this is really the only novel result. As such, I feel it deserves special scrutiny, and for me, it doesn’t hold up to my satisfaction. I just wanted to see if anyone else thought the same, or if I was just being too critical.


  6. oops    11 April 08    #

    it’s “Shine-Dalgarno,” not “-Delgarno” fyi.

    also PhillipJ, it’s a little disingenuous to say the authors described the codon stepsize as “striking,” don’t you think? a careful read will show that they were referring to the discontinuous start-stop-start nature of translocation…

    spooner, one can certainly say is that the distribution isn’t a single exponential… but whether it’s two or three sequential steps……………?


  7. Philipj    11 April 08    #

    oops — thanks, fixed.

    I don’t think what I said was disingenuous at all, as I’m not just talking about the codon stepsize, but the traces entirely. Given that all of these experiments (whether it be RNAp, kinesin, etc) show these kinds of traces when the kinetics are slow enough to be resolved (and the instrument is of sufficiently high resolution), it is less striking and more expected.


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