A Passage Through the Ribosome by Cryo-EM - Biophysical Approaches to Translational Control of Gene Expression - page 31

and recycling process, and for the reason of simplicity, here we are describing mainly
the concepts that were learnt from studying the bacterial translation system.

2.2.1 Initiation

In bacteria, with the help of initiation factors IF1, IF2, and IF3, an fMet-tRNA fMet
binds to the correct messenger RNA start codon on the 30S. Thus the 30S initiation
complex (30SIC) is formed, which is then joined by the 50S to form the 70S initia-
tion complex (70SIC) followed by subsequent release of the initiation factors.
Following three-dimensional multivariate statistical analysis and three-dimensional
classification into structural sets with low intraclass of three-dimensional recon-
structions from randomly selected particle images, Simonetti et al. ( 2008 ) reported
a detailed description of the 30SIC formation, containing mRNA, fMet-tRNA fMet ,
and IF1 and IF2

GTP densities. The study revealed characteristic and precise posi-
tioning of the fMet-tRNA fMet where it is held stable on the 30S. Its anticodon end is
anchored to the decoding site of the 30S while its aminoacyl end is bound to the IF2.
This observation thus helped explain the rate-limiting step in translation, i.e., the
basis of the formation of a stable 30SIC.
First insights into the 70SIC also came from two cryo-EM studies by Allen et al.
( 2005 ) and Myasnikov et al. ( 2005 ). Together these studies shed lights on the mech-
anism of IF2-mediated fMet-tRNA fMet positioning to the peptidyl/initiation (P/I) site
of the ribosome. Analyses from the 70SICs complex comprising the mRNA, fMet-
tRNA fMet , and IF2 with either a non-hydrolyzable GTP analog or GDP show sub-
stantial conformational changes of the IF2 and of the entire ribosome. It also reveals
that the GTP binding domain of IF2 interacts with the GTPase-associated center of
the 50S subunit in a mode similar to Elongation Factor-G (EF-G) and Elongation
Factor-Tu (EF-Tu). In the non-hydrolyzable GTP-bound state, IF2 interacts mostly
with the small subunit and positions the fMet-tRNA fMet to the P/I site, whereas in the
GDP-bound state IF2 adopts a more compact conformation suitable for release from
the ribosome. It also provided insights into the molecular mechanism for release of
IF1 and IF3.

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2.2.2 Elongation Cycle

Extensive cryo-EM studies have helped understand this process which is mainly the
mechanism in which the polypeptide chain grows by one amino acid following each
elongation cycle that consists of the following steps, like appropriate aminoacyl-
tRNA selection and accommodation, peptidyl transferase reaction between the
P-site tRNA polypeptide-chain and A-site aa-tRNA, followed by tRNA-mRNA transloca-
tion by one codon towards the 3¢ mRNA direction, and subsequent release of an
deacylated tRNA from the E site of the ribosome. Two elongation factors EF-Tu and