Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 132

Fig. 7.4 Ribosome recycling. ( a ) Subunit dissociation by RRF, EF-G, and IF3 in the presence and
absence of fusidic acid. Changes in light scattering at 435 nm were monitored (arbitrary units, a.u.)
(Savelsbergh et al. 2009 ) . ( b ) RRF-mediated delay of Pi release from EF-G (Savelsbergh et al.
2009 ) . ( c ) Sequence of steps in post-termination complex disassembly. The ribosomal subunits are
depicted in light gray (50S) and dark gray or pale lilac (30S) depending on the conformation.
EF-G is shown in pink , red , and yellow in the GTP-, GDP·Pi- and GDP-bound forms, respectively.
RRF is in cyan , IF3 in magenta , and tRNA in green
Savelsbergh et al. 2005, 2009 ); in fact, GTP hydrolysis by EF-G is independent of
the functional state of the ribosome and seems to be required to stabilize EF-G bind-
ing to the ribosome (Wilden et al. 2006 ). Pi release is delayed, albeit to a somewhat
different extent during translocation (about 15 ms) compared to recycling (30 ms).
However, there are also important differences between the two reactions, indicating
that the coupling of the energy of EF-G binding and GTP hydrolysis is different.
When GTP hydrolysis is prevented by replacing GTP with non-hydrolyzable ana-
logs, translocation still takes place, albeit slowly (Katunin et al. 2002b ; Rodnina et al.
1997 ), whereas ribosome disassembly by EF-G/RRF is completely blocked (Karimi
et al. 1999 ; Peske et al. 2005 ; Zavialov et al. 2005 ). Vanadate, an analog of Pi, strongly
impairs recycling, but has no effect on translocation (Savelsbergh et al. 2009 ) . Fusidic
acid, which is a known inhibitor of EF-G function, stalls a particular EF-G conforma-
tion which in the absence of the antibiotic is short-lived. Upon translocation, this