Seminar for PhD students - Max-Planck-Institut für biophysikalische ...

Jörg Mittelstät
Physical Biochemistry
Organizing team: W. Fischle, G. Groenhof, C. Höbartner, S. Jakobs, A. Lange
Induced fit in biological recognition: GTPase activation of
translation elongation factor Tu on the ribosome
The ribosome synthesizes proteins from
aminoacyl-tRNAs (aa-tRNAs) based on the
sequence of codons of an mRNA template.
During decoding, aa-tRNA is delivered to
the ribosome in a very tight complex with
elongation factor Tu (EF-Tu) and GTP. The
release of aa-tRNA from EF-Tu requires
GTP hydrolysis which, in turn, is allowed
only when codon recognition has taken
place. The mechanism of coupling is induced
fit. After initial binding, the anticodon
of the aa-tRNA in the complex with
EF-Tu probes the mRNA codon presented
in the decoding site of the ribosome
(Fig. 1). When the anticodon matches the
codon, rapid GTP hydrolysis is triggered,
which is an important checkpoint for tRNA
selection. If the codon-anticodon duplex
contains mismatches, activation of GTP
hydrolysis is very slow which allows the
ribosome to reject the incorrect substrate.
How codon recognition is signaled from
the decoding center to the GTP binding
pocket of EF-Tu, which is located more
than 75 Å away, is not understood. Transient
distortions of the aa-tRNA were suggested
to have an active role in signaling
and in this study we tested this hypothesis.
Transient conformational changes
of tRNA during binding of the EF-Tu
GTP·aminoacyl-tRNA complex can be followed
in a stopped-flow apparatus monitoring
the fluorescence intensity changes
of a proflavin label attached to aa-tRNA.
When a codon complementary to the
aa-tRNA is found in the A site, the tRNA assumes
a short-lived conformation of higher
fluorescence (Fig. 1A), which is due to the
distortion of the tRNA molecule. The higher
sensitivity of the fluorophore against
quenching by potassium iodide (Fig. 1A)
indicates that the tRNA molecule in the
transient state has a somewhat more open
conformation. A similar distortion is observed
also upon aa-tRNA binding at a
near-cognate codon (Fig. 1B and C), however,
only for a very small fraction of the
tRNA molecules, which correlates with
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Seminar
Seminar for
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21.10.2009
17:00 c.t.
Large seminar room
the level of misreading at the initial selection
step, i.e. before GTP is hydrolyzed.
Our data favors a model in which tRNA
distortion is required for the GTPase activation
in EF-Tu, and is involved in communicating
the signal from the decoding site
to the factor. Aa-tRNA enters the decoding
site and samples the mRNA codon in
a non-distorted conformation; formation
of the cognate codon-anticodon complex
induces conformational changes of the
ribosome, aa-tRNA and EF-Tu resulting
in the activation of GTP hydrolysis by
EF-Tu – an example of how induced fit can
be used for substrate selection.
References for further reading:
Rodnina, M.V. (2009a) Long-range signalling in
activation of the translational GTPase EF-Tu.
EMBO J 28: 619-620.
Rodnina, M. V. (2009b) Visualizing the protein
synthesis machinery: New focus on the translational
GTPase elongation factor Tu. Proc Natl
Acad Sci USA 106: 969-70.
Schuette, J.-C., Murphy, F. V., Kelley, A. C., Weir,
J. R., Giesebrecht, J., Connell, S. R., Loerke, J.,
Mielke, T., Zhang, W., Penczek, P. A., Ramakrishnan
V., and Spahn, C. M. T. (2009) GTPase
activation of elongation factor EF-Tu by the ribosome
during decoding. EMBO J 28: 755-765.
Villa, E., Sengupta, J., Trabuco, L. G., LeBarron,
J., Baxter, W. T., Shaikh, T. R., Grassucci, R.
A., Nissen, P., Ehrenberg, M., Schulten, K. and
Frank, J. (2009) Ribosome-induced changes
in elongation factor Tu conformation control
GTP hydrolysis. Proc Natl Acad Sci USA 106:
1063-1068.
Fig. 1: A. Sequence of steps during GTPase activation of EF-Tu on the ribosome (modified from Rodnina et al., 2009a). E, P and A denote the three
tRNA binding sites of the ribosome. GTP* denotes the GTPase-activated conformation of EF-Tu. Distortion of the tRNA molecule is indicated by a kink.
B-D. Distortion of the aa-tRNA during A-site binding monitored by transient fluorescence quenching. B) Cognate codon. C) Near-cognate codon with
a single mismatch in the codon-anticodon duplex. The topmost trace (dark blue) is the unquenched signal while the lower traces (blue to light blue) are
obtained by increasing the concentration of potassium iodide (KI) in the reaction. D) Analysis of the differential amplitudes of the high-fluorescence intermediates
according to the Stern-Volmer equation in the following form: A 0 - A = A0(1-1/(1+K SV[KI])), where A 0-A is the amplitude of the fluorescence
increase determined from differential curves (not shown), yields quenching constants (K SV) of 11 ± 3 M -1 for the cognate and 22 ± 6 M -1 for the nearcognate
codon. Compared to a quenching constant of 5 ± 0.1 M -1 determined for the free tRNA, this indicates a distorted, more open conformation of
the tRNA in both cases. However, only a small fraction of the tRNA molecules undergoes the distortion at a near-cognate codon.