A Passage Through the Ribosome by Cryo-EM - Biophysical Approaches to Translational Control of Gene Expression - page 43

because in in vivo , a large protein complex (eIF4F) is bound to the 5¢ end of the
mRNA. The latch is proposed to clamp around the mRNA, either after initiation
codon recognition or after subunit joining, to trap the mRNA on the ribosome, pre-
venting mRNA dissociation. eIF1 is hypothesized to indirectly monitor initiation
codon recognition by influencing the conformation of the platform and the positions
of mRNA and tRNA. eIF1A is expected to bind in a similar position to IF1. Both the
eIF1 and eIF1A are required for the full conformational change, and the change is
fundamentally required for translation initiation (Passmore et al. 2007 ) . Similar
kind of conformational change in 40S subunit is also shown in a 43S complex of
40S and multifactor complex (eIF1, eIF1A, eIF3, and eIF5) in budding yeast saying
that this conformational change in 40S subunit leads to opening of head/body/plat-
form junction, thus opening the mRNA entry channel (Gilbert et al. 2007 ) .
The second mode of translation initiation is by IRES, present in the upstream of
few cellular mRNA and in viral genomic RNAs. They manipulate the translation
system in such a manner that even in the absence of translation initiation factors
they begin the translation of mRNA containing IRES in their upstream region. This
kind of translation initiation mechanism is seen in viral mRNA and also in few
mRNA expressing during stress conditions when the normal cellular translation is
stopped. IRES can bind directly to the 40S subunit and can bypass the initiation
process directly proceeding to the elongation step. Some of the IRES of viruses do
not require any translation initiation factor and not even initiator tRNA like Cricket
paralysis virus (CrPV). But few of the viral IRES require some of the initiation fac-
tors like Hepatitis C virus (HCV) requires eIF2, eIF3, and Met-tRNA and polio
virus requires eIF4E (Ji et al. 2004 ). Through cryo-EM, translation initiation by two
IRES of HCV and CrPV was studied. In HCV, IRES consist of three domains;
domain II, domain III, and domain IV containing the AUG start codon. The HCV
IRES binds to the solvent side of the 40S subunit, where domain II interacting with
S5 which forms the mRNA exit channel along with S14 and the apex of the domain
II binds close to the tRNA E site. Domain III is mainly involved in binding of 40S
subunit and eIF3 (Berry et al. 2011 ). These interactions induce a conformational
change in the 40S subunit which is required for placing the start codon at appropri-
ate position in P site to initiate translation and also 60S subunit binding. The confor-
mational changes induced in the 40S are similar to the conformation changes which
take place upon binding of eIFs to the 40S in normal translation initiation. To pre-
cede to the elongation step, the domain II should be removed from the E site during
the first translocation step, that is the reason why IRES translation initiation is more
sensitive to cyclohexamide than normal translation initiation as they bind to the E
site and prevent the removal of domain II from the E site (Boehringer et al. 2005 ) .
The mechanism by which the IRES of CrPV initiate translation is a bit different
from that of HCV. CrPV IRES contains three psuedoknots; PK1 which binds to the
P site, PK2 and PK3 which binds to the E site. PK1 mimics the initiator tRNA, that
is the reason why CrPV IRES do not require even initiator tRNA to initiate transla-
tion and the binding of PK1 to the P site positions the first codon to the A site. As it
was seen in the HCV the binding of IRES of CrPV also induces a conformational
change in the 40S subunit, thus preparing the ribosome to proceed for the further steps.