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the fully ratcheted conformation, which was also described in later X-ray studies of
E. coli
ribosome (Dunkle et al.
2011
) .
When compared to the structure of the unratcheted prokaryotic ribosome (Jenner
et al.
2010b
) our model of the eukaryotic ribosome shows a 5° counter-clockwise
rotation of the 40S subunit body relative to the 60S subunit and a swiveling of the
40S head domain by 14° in the direction of the E-site tRNA (Fig.
1.9a
, b). These
characteristics are in agreement with cryo-EM observations demonstrating that
vacant yeast ribosomes assume a ratcheted conformation similar to the one stabi-
lized by the binding of eukaryotic elongation factor 2 (eEF2) (Spahn et al.
2004a
) .
Indeed, our X-ray model fits well into the cryo-EM maps of the 80S-eEF2 complex
(data not shown).
As a result of the large-scale movements implicated in ratcheting there are
significant alterations in the bridges between the head domain of the small subunit
and the large subunit, in comparison with the unratcheted prokaryotic ribosome
(Fig.
1.9c-f
). In the latter, the first bridge between the small subunit head domain
and the large subunit, bridge B1a, is formed by the A-site finger (H38 of 23S) and
protein S13 (Fig.
1.9d
). Since head swiveling displaces components at the periphery
of the head domain by as much as 25 Å, this bridge is rearranged in our model
(Fig.
1.9c
, e). We find that ratcheting brings residues 1,239-1,241 at the tip of h33 (a
component of the beak of 40S) as well as protein S15 (prokaryotic homolog, S19p),
into proximity of the tip of H38 that bends significantly in order to form interactions
with these partners. Conformational changes are also observed at the base of H38
where it contacts the central protuberance. In the second bridge between the head
domain of 40S and the 60S subunit, B1b (Fig.
1.9c
, f), the large shift in the position
of protein S18 (S13p) places its largest helix, instead of the N-terminal loop, in
contact with protein L11 (L5p) of the central protuberance. In addition, residues
from loop 65-75 in S15 (S19p) may also interact with L11 (L5p) in the ratcheted
state. The prokaryotic homologues of the two proteins, S15 (S19 p) and S1 (S13p),
were shown to monitor the occupancy of the A and P sites in the non-ratcheted state
(Jenner et al.
2010b
). In eukaryotes these two proteins probably interact stronger
than in prokaryotes because they have significant eukaryote-specific extensions in
the interacting area.
The extent of the head's rotation may be limited or determined by numerous
weak interactions between the head and the large subunit. Hence, multiple weak
interactions facilitate a wide but precise ratcheting movement. The observed
flexibility of the interacting partners is probably crucial for constantly adjusting the
bridges as the ratcheting movement progresses.
Fig. 1.9
(continued) (
b
) View from the solvent side of 40S. (
c
) The bridge B1 in the ratcheted
80S. (
d
) Bridge 1 of the non-ratcheted prokaryotic ribosome. (
e
)
Close-up view
of bridge B1a.
The tip of the A-site finger (ASF-H38) from 25S RNA forms interactions (colored in
red
) with the
head of the 40S subunit including protein S15 (
magenta
). (
f
) View of bridge B1b formed between
proteins S18 and protein L11. Residues thought to interact are indicated in
red
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