[tt] softmachines: timing of translation by ribosome

[Alejandro Dubrovsky]

(
http://www.softmachines.org/wordpress/?p=398
and abstract:
http://www.nature.com/nature/journal/v452/n7187/abs/nature06716.html
)


Watching an assembler at work

The only software-controlled molecular assembler we know about is the
ribosome - the biological machine that reads the sequence of bases on a
strand of messenger RNA, and, converting this genetic code into a
sequence of amino acids, synthesises the protein molecule that
corresponds to the gene whose information was transferred by the RNA. An
article in this week’s Nature (abstract, subscription required for full
paper, see also this editor’s summary) describes a remarkable
experimental study of the way the RNA molecule is pulled through the
ribosome as each step of its code is read and executed. This
experimental tour-de-force of single molecule biophysics, whose first
author is Jin-Der Wen, comes from the groups of Ignacio Tinoco and
Carlos Bustamante at Berkeley.

The experiment starts by tethering a strand of RNA between two
micron-size polystyrene beads. One bead is held firm on a micropipette,
while the other bead is held in an optical trap - the point at which a
highly focused laser beam has its maximum intensity. The central part of
the RNA molecule is twisted into a single hairpin, and the ribosome
binds to the RNA just to one side of this hairpin. As the ribosome reads
the RNA molecule, it pulls the hairpin apart, and the resulting
lengthening of the RNA strand is directly measured from the change in
position of the anchoring bead in its optical trap. What’s seen is a
series of steps - the ribosome moves about 2.7 nm in about a tenth of a
second, then pauses for a couple of seconds before making another step.

This distance corresponds exactly to the size of the triplet of bases
that represent a single character of the genetic code - the codon. What
we are seeing, then, is the ribosome pausing on a codon to read it,
before pulling the tape through to read the next character. What we
don’t see in this experiment, though we know it’s happening, is the
addition of a single amino acid to the growing protein chain during this
read step. This takes place by means of the binding to RNA codon, within
the ribosome, of a shorter strand of RNA - the transfer RNA - to which
the amino acid is attached. What the experiment does make clear that the
operation of this machine is by no means mechanical and regular. The
times taken for the ribosome to move from the reading position for one
codon to the next - the translocation times - are fairly tightly
distributed around an average value of around 0.08 seconds, but the
dwell times on each codon vary from a fraction of a second up to a few
seconds. Occasionally the ribosome stops entirely for a few minutes.

This experiment is far from the final word on the way ribosomes operate.
I can imagine, for example, that people are going to be making strenuous
efforts to attach a probe directly to the ribosome, rather than, as was
done here, inferring its motion from the location of the end of the RNA
strand. But it’s fascinating to have such a direct probe of one of the
most central operations of biology. And for those attempting the very
ambitious task of creating a synthetic analogue of a ribosome, these
insights will be invaluable.

---

Nature 452, 598-603 (3 April 2008) | doi:10.1038/nature06716; Received 4
September 2007; Accepted 16 January 2008; Published online 9 March 2008

Following translation by single ribosomes one codon at a time

Jin-Der Wen1, Laura Lancaster2, Courtney Hodges3, Ana-Carolina Zeri4,
Shige H. Yoshimura5, Harry F. Noller2, Carlos Bustamante1,3,6 & Ignacio
Tinoco1

 1. Department of Chemistry, University of California, Berkeley,
California 94720, USA
 2. Department of Molecular, Cell, and Developmental Biology, and Center
for Molecular Biology of RNA, University of California, Santa Cruz,
California 95064, USA
 3. Biophysics Graduate Group, University of California, Berkeley,
California 94720, USA
 4. Brazilian Synchrotron Light Laboratory, Caixa Postal 6192, Campinas
SP 13083-970, Brazil
 5. Graduate School of Biostudies, Kyoto University, Yoshida-honmachi,
Sakyo-ku, Kyoto, 606-8501, Japan
 6. Howard Hughes Medical Institute, Department of Physics and Molecular
and Cell Biology, University of California, Berkeley, California 94720,
USA 

Correspondence to: Ignacio Tinoco1 Correspondence and requests for
materials should be addressed to I.T. (Email: intinoco at lbl.gov).

Top of page
Abstract

We have followed individual ribosomes as they translate single messenger
RNA hairpins tethered by the ends to optical tweezers. Here we reveal
that translation occurs through successive translocation-and-pause
cycles. The distribution of pause lengths, with a median of 2.8 s,
indicates that at least two rate-determining processes control each
pause. Each translocation step measures three bases—one codon—and occurs
in less than 0.1 s. Analysis of the times required for translocation
reveals, surprisingly, that there are three substeps in each step. Pause
lengths, and thus the overall rate of translation, depend on the
secondary structure of the mRNA; the applied force destabilizes
secondary structure and decreases pause durations, but does not affect
translocation times. Translocation and RNA unwinding are strictly
coupled ribosomal functions.