A Passage Through the Ribosome by Cryo-EM - Biophysical Approaches to Translational Control of Gene Expression - page 34

a large movement of the G¢ domain of the EF-G associated with the GTP hydrolysis.
Most importantly this study for the first time revealed structural evidence of the
involvement of the highly mobile C-terminal domain of ribosomal protein L7/L12
in the translation process by revealing molecular interaction of the CTD of L7/L12
with the G¢ domain of the EF-G in GDP state. This observation was later supported
by other combined X-ray crystallographic and cryo-EM (Harms et al. 2008 ) and
X-ray crystallographic studies (Gao et al. 2009 ). Using combined X-ray crystallo-
graphic and cryo-EM approach Connell et al. ( 2007 ) revealed a detailed insight into
the ribosome-dependent GTPase activity of EF-G during the translocation process.
It showed that the dynamic reorganization of the functionally important, P loop,
switch I and II regions of EF-G which interact with the bound GTP, occurred upon
EF-G's interactions with the ribosome during the ratcheting motions.
Our understanding on the pathways of tRNA translocation on the ribosome has
been advanced further by finding of tRNAs in distinct hybrid states (A/P and P/E)
in spontaneously ratcheted 70S-tRNA complexes (Agirrezabala et al. 2008 ) . Most
recently, using multiparticle cryo-EM analysis Ratje et al. ( 2010 ) reported direct
structural and mechanistic insight into the previously unseen tRNA intermediates
involved in the universally conserved translocation process. They found two previ-
ously unseen subpopulations of translocation complexes having a novel intra-
subunit tRNA in pe/E hybrid state that is stabilized by domain IV of EF-G, which
interacts with the swiveled 30S-head conformation thus advancing the field of
research further.

2.2.3 Termination

When the translating ribosome reaches to a stop codon UAA, UAG, and UGA mov-
ing into the ribosomal A site, the elongation cycle stops due to the nonavailability of
a corresponding tRNA. Instead, depending on the nature of the stop codons, either
one of the class I release factors, RF1 or RF2, binds to the decoding center of the
ribosome, and triggers the hydrolysis of the ester bond that links the polypeptide
with the P-site tRNA, thereby facilitating the release of the polypeptide from the
ribosome. Much of the structural and functional insights of the above phenomenon
came from several cryo-EM studies. Rawat et al. ( 2006 ) visualized ribosome-bound
RF1 is in an open conformation, unlike the closed conformation observed in the
crystal structure of the free factor. Similar results were obtained previously for RF2
study (Rawat et al. 2003 ; Klaholz et al. 2003 ). The open form of RF1 and RF2
allowed their simultaneous access to both the decoding center and the peptidyl-trans-
ferase center (PTC) on the ribosome. By connecting the ribosomal decoding center
with the PTC through two conserved motifs, RF1 and RF2 functionally mimic a tRNA
molecule in the A site; this showed another example of molecular mimicry as was seen
previously between the EF-G and the TC during the elongation event. Finally RF3,