A Passage Through the Ribosome by Cryo-EM - Biophysical Approaches to Translational Control of Gene Expression - page 32

A

a b c

A

/

T

ASL

Anticodon
arm

Fig. 2.3 Model of A/T-site tRNA and its interaction with ribosome. ( a ) Rendering of the fitted
tRNA Phe inside the cryo-EM density for the A/T-site tRNA. Evidently, the anticodon arm of the
X-ray structure does not fit the features of the density for the A/T-site tRNA. ( b ) Modeled structure
for the A/T-tRNA is represented in green ribbons, showing an evident improvement in the docking
of the coordinates within the density. ( c ) Comparison of the modeled coordinates for the A/T-site
tRNA ( green ) and the coordinates for the A-site tRNA ( gold ). (The docking of the A-site tRNA in
this cryo-EM density did not require any modification in the anticodon arm of the X-ray structure.)
The Anti-codon Stem Loops (ASLs) of both tRNAs are similarly oriented. Source : Fig 4 from the
following reference Valle et al. ( 2003a, b )

EF-G play major role here. Structural and functional observations from relevant
cryo-EM studies on bacteria are described below.

2.2.2.1 tRNA Selection and Accommodation

After the initiation step, an aminoacyl-tRNA is brought to the ribosome by EF-Tu
for delivery of the next amino acid to the growing polypeptide chain. This is a cru-
cial step where only that aa-tRNA which has the correct nucleotides at its anticodon
end against the specific codon sequence in the translating mRNA will be selected
and then accommodated into the ribosome for the subsequent peptidyl transferase
and translocation process. Fairly detailed mechanisms underlying this important
aspect were first revealed by cryo-EM by studying various 70S-Ternary Complexes
(70S

GTP + Kirromycin; and its derivative complexes].
Results from several of those studies (Stark et al. 2002 ; Valle et al. 2002 ; 2003a, b ;
Li et al. 2008 ; Schuette et al. 2009 ; Villa et al. 2009 ) depicted that the EF-Tu-bound
incoming cognate aa-tRNA (the CCA end of the aa-tRNA is attached to the EF-Tu,
whereas anticodon end is free) possess a partially bended conformation at its anti-
codon end, compared to the X-ray structure of free-standing tRNA, (see Fig. 2.3 )
during the initial steps of aa-tRNA selection prior to the EF-Tu

⋅ TC), [70S ⋅ EF-Tu ⋅ aa-tRNA ⋅

GDP is released.
This partial bending in a suitable angle allows interaction between the anticodon of
the tRNA and the nucleotides of the translating mRNA in the decoding center while
that particular aa-tRNA is still attached to the EF-Tu; thus screening for the cognate
tRNAs are facilitated. After the cognate tRNA is recognized, a series of conforma-
tional changes take place on the associated ribosomal components and the EF-Tu,
GTP hydrolysis occurs, and the EF-Tu releases the CCA end of the aa-tRNA and
goes out of the ribosome. Soon after the cognate tRNA firmly establishes its interac-
tion with the codon (referred as the A/T state), and gets released from the EF-Tu, the
deformity in the anticodon arm is relieved. Mechanical energy released from this, in

⋅