ayout 1 - EMBL Grenoble

EMBL Research at a Glance 2009
Christiane
Schaffitzel
PhD 2001, University of
Zürich.
Postdoctoral research at the
Institute of Molecular Biology
and Biophysics, ETH Zürich.
Team leader at EMBL
Grenoble since 2007.
Habilitation, ETH Zürich,
2008.
Cryo-EM of ribosomal complexes
Previous and current research
How exactly the folding of a nascent polypeptide chain into its native structure is achieved is one
of the fundamental questions in biology. Synthesis and folding of proteins require concerted interactions
of the translating ribosome with translation factors, processing enzymes, molecular
chaperones and factors involved in the export of proteins. The functional and structural characterisation
of such complexes is the aim of our team.
We use cryo-EM and single particle analysis to study nascent chain co-translational folding, targeting
and translocation. Structures of translating ribosomes in complex with the factors involved
provide important mechanistic insight into the molecular mechanism of protein sorting.
A prerequisite for any structural studies on translating ribosomes is to produce large amounts of homogenous,
stable complexes. To this end, we adapted and decisively improved existing E. coli in
vitro translation protocols which are optimised for protein expression for the production of ribosome
nascent chain complexes. We have solved the structures of a translating ribosome-signal
recognition particle (SRP) complex and of a non-translating ribosome without nascent chain in
complex with SRP. Conformational changes of SRP which occur when SRP recognises a signal sequence
on the nascent chain emerging from the ribosomal exit tunnel explain previous biochemical
findings such as experimental crosslinks, salt-sensitivity of the 70S-SRP complex and a more
than 100-fold improved affinity of SRP towards ribosomes displaying a nascent chain with a signal
sequence. Furthermore, the structure of a translating ribosome in complex with the E. coli translocation machinery at 15 Å resolution reveals
that both active and inactive translocon are composed of dimers of SecYEG. Cryo-EM analysis of a ribosome in complex with the molecular
chaperone Trigger Factor suggests that Trigger Factor forms a hydrophobic arch over the ribosomal exit tunnel, thereby providing a folding interface
for the nascent polypeptide and protection against other cytosolic proteins to prevent aggregation and degradation of nascent proteins.
Our findings in aggregate support a view of the ribosomal exit tunnel as an anchor point for a multitude of factors involved in co-translational
processes. A concerted action of these factors ensures correct processing, folding and targeting of nascent proteins.
Future projects and goals
In the future, we will continue analysing ribosomal complexes involved in co-translational processes to gain structural insight into all steps
along the pathway of co-translational targeting and translocation.
In collaboration with the Berger group (page 89), we study human macromolecular complexes involved in class II gene expression. We use
EM to analyse the structure of these molecular machines, which we produce by means of advanced recombinant technologies.
Figure 1: Cryo-EM
structure of the E. coli
signal recognition
particle bound to a
translating ribosome.
Ribosomal subunits are
shown in yellow (30S)
and blue (50S), tRNAs in
green and orange, the
SRP in red. Below the
atomic model of E. coli
SRP as fitted into the
density is depicted.
Figure 2: EM reconstruction of the
ribosome nascent chain complex
and the translocon. The 30S
ribosomal subunit is shown in
yellow, the 50S subunit in light
blue, the A, P and E site tRNAs are
in magenta, green and orange
respectively. The mRNA is
depicted in cyan and the nascent
chain in the polypeptide exit tunnel
in green. The active translocon in
dark blue is located at the exit of
the ribosomal tunnel. A second,
non-translocating translocon (red)
is bound to the mRNA.
Selected references
Zhang, X., Schaffitzel, C., Ban, N. & Shan, S.O. (2009). Multiple
conformational switches in a GTPase complex control cotranslational
protein targeting. Proc Natl Acad Sci. USA, 106, 175-9
Bingel-Erlenmeyer, R., Kohler, R., Kramer, G., Sandikci, A., Antolic,
S., Maier, T., Schaffitzel, C., Wiedmann, B., Bukau, B. & Ban, N.
(2008). A peptide deformylase-ribosome complex reveals
mechanism of nascent chain processing. Nature, 52, 108-11
96
Schaffitzel, C., Oswald, M., Berger, I., Ishikawa, T., Abrahams, J.P.,
Koerten, H.K., Koning, R.I. & Ban, N. (2006). Structure of the E. coli
signal recognition particle bound to a translating ribosome. Nature,
, 503-506
Mitra, K., Schaffitzel, C., Shaikh, T., Tama, F., Jenni, S., Brooks CL,
3rd, Ban, N. & Frank, J. (2005). Structure of the E. coli proteinconducting
channel bound to a translating ribosome. Nature, 38,
318-32