To provide a bridge between in vivo and in vitro studies of
eukaryotic translation initiation, we have developed a
reconstituted translation initiation system using components
from the yeast Saccharomyces cerevisiae. We have purified
a minimal set of initiation factors (eIFs) that, together with
yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are
sufficient to assemble active initiation complexes on a minimal
mRNA template. The kinetics of various steps in the pathway
of initiation complex assembly and the formation of the first
peptide bond in vitro have been explored. The formation of active
initiation complexes in this system is dependent on ribosomes,
mRNA, Met-tRNAi, GTP hydrolysis, eIF1, eIF1A, eIF2,
eIF5, and eIF5B. Our data indicate that eIF1 and eIF1A both
facilitate the binding of the
eIF2[bull ]GTP[bull ]Met-tRNAi complex to the 40S
ribosomal subunit to form the 43S complex. eIF5 stimulates a
step after 43S complex formation, consistent with its proposed
role in activating GTP hydrolysis by eIF2 upon initiation codon
recognition. The presence of eIF5B is required for the joining
of the 40S and 60S subunits to form the 80S initiation complex.
The step at which each of these factors acts in this reconstituted
system is in agreement with previous data from in vivo studies
and work using reconstituted mammalian systems, indicating that
the system recapitulates fundamental events in translation
initiation in eukaryotic cells. This system should allow us
to couple powerful yeast genetic and molecular biological
experiments with in vitro kinetic and biophysical experiments,
yielding a better understanding of the molecular mechanics of
this central, complex process.