download report - Istituto Pasteur

P a r t i c i p a n t s :
Dario Benelli, research fellow; Antimo Naspi, PhD student;
Dorina Polinari, undergraduate student.
C o l l a b o r a t i o n s :
University of Wien, Austria (Prof. Udo Blaesi); University J.W.
Goethe, Frankfurt, Germany (Prof. Joerg Soppa); CNRS,
Strasbourg, France (Dr. Bruno Klaholtz); Università Politecnica
delle Marche, Ancona (Prof. Anna La Teana).
Report of activity
It is well known that the process of gene expression
in Archaea has many features in common with that of
Eukarya. This is especially true for the initiation step
of translation, which in Archaea and in Eukarya has
a similar high complexity. As the speed and efficiency
of translation are modulated mainly at the initiation
stage, the origin of the regulatory mechanisms
acting in present-day cells may be traced back to the
common ancestor of Archaea and Eukarya.
Therefore, unravelling the mechanism and regulation
of translational initiation in Archaea, besides
being interesting in its own right, will also lead to a
better understanding of the corresponding eukaryal
process.
The project is specifically centred on investigating
the function and mechanism of action in Archaea of
two translation initiation factors, a/eIF2 and aIF6,
shared by the Archaea and the Eukarya but absent in
Bacteria. The eukaryal factors (eIF2 and eIF6) have
important roles in regulating protein synthesis in
both physiological and pathological situations, but
several aspects of their function remain to be clarified.
The function of a/eIF2 and aIF6 in Archaea is
currently poorly understood; its elucidation will
help to trace a meaningful picture of the early evolution
of these interesting proteins, thus gaining
valuable insight into the onset of eukaryotic-type
translational regulation.
11
Molecular biology of microorganisms and viruses - AREA 1
Translational regulation: from the archaea to the eukarya
Principal investigator: Paola Londei
Professor of Applied Biology
Dipartimento Biotecnologie Cellulari ed Ematologia
Tel: (+39) 06 4940463; Fax: (+39) 06 4462891
londei@bce.uniroma1.it
An important regulator of translation: a/e IF2
In both Archaea and Eukarya, a/eIF2 is trimeric
protein consisting of the α, β and γ subunits. It interacts
with initiator tRNA (met-tRNAi) and delivers it
to the ribosome. It is a G-protein, active only in the
GTP-bound form. In eukaryotes, after delivering
met-tRNAi, eIF2 hydrolyzes its GTP and is ejected
from the ribosome in the inactive, GDP-bound form.
To participate in another round of initiation, eIF2
must be reactivated by GTP/GDP exchange,
catalyzed by the auxiliary factor eIF2B. In most conditions
(such as stress) when a rapid shut-off of protein
synthesis is desirable, eIF2 is inactivated by
phosphorylation (at ser 51) of its α-subunit, carried
out by certain stress-activated kinases. This modification
converts the α-protein in a competitive
inhibitor of eIF2B, thereby inhibiting GTP/GDP
exchange and blocking the recycling of eIF2. This
mechanism of translational control is essential and
widespread in eukaryotic cells, but does not exist in
Bacteria, which use a different factor, the monomeric
protein IF2, for delivering tRNAi to the ribosome.
Why the Archaea, as Eukarya, should use a trimeric
protein as the met-tRNAi binding factor is as yet
unclear. a/eIF2 cannot be regulated with the same
mechanism as in eukaryotes; it has a similar affinity
for both GTP and GTP and does not need an
exchange factor to be reactivated after GTP hydrolysis.
Since it has been reported that the α-subunit of
a/eIF2 is phosphorylated as in eukarya, one of the
aims of our project is to verify this finding and investigate
the physiological role of α-subunit modifications
in Archaea.
The modification pattern of the a/eIF2 α-subunit in
the archaeon Sulfolobus solfataricus is studied both in
vitro and in vivo. We have cloned, expressed and
purified an archaeal kinase reported to be involved
in a/eIF2 phosphorylation. The recombinant protein
efficiently phosphorylates the a/eIF2 α-subunit
in vitro. The modified a/eIF2 α-subunit does not
seem to differ from its native counterpart in a series