Rapid Kinetic Analysis of Protein Synthesis - Biophysical Approaches to Translational Control of Gene Expression - page 131

In the presence of EF-G, the directionality of tRNA movement is biased in such
a way that in the post-translocation state domain 4 of EF-G—which is crucial for
translocation (Rodnina et al. 1997 ; Savelsbergh et al. 2000 ) —occupies the 30S A
site (Agrawal et al. 1998 ; Frank and Agrawal 2000 ; Stark et al. 2000 ) , thus effec-
tively preventing back movement of peptidyl-tRNA while the unlocked state pre-
vails. On EF-G, an insertion in the GTP binding domain, the G¢ subdomain, appears
to be important for EF-G binding to the ribosome and the conformational coupling
between GTP hydrolysis, retention of Pi, and unlocking (Agrawal et al. 2001 ;
Nechifor et al. 2007 ; Ticu et al. 2009 ) .

7.6.2 Ribosome Recycling

The dissociation of the ribosome into subunits following termination (ribosome
recycling) is promoted by the RRF and EF-G. The reaction provides an example
where the reaction was monitored in stopped-flow utilizing the decrease of light
scattering intensity upon dissociation of the 70S ribosome into 30S and 50S sub-
units (Fig. 7.4a ).

7.6.2.1 Sequence of Events

RRF and EF-G·GTP are recruited to the post-termination complex with the deacy-
lated tRNA bound to the hybrid P/E state (Lancaster et al. 2002 ; Peske et al. 2005 ) .
Interactions between RRF and EF-G on the ribosome are crucial for the catalysis of
ribosome splitting (Gao et al. 2007 ; Ishino et al. 2000 ). GTP hydrolysis by EF-G in
the recycling reaction is rapid, comparable to that during translocation (Savelsbergh
et al. 2009 ). However, the presence of RRF delays Pi release by about 30 ms
(Fig. 7.4b ), suggesting that the complex undergoes a rearrangement that controls the
following Pi release. In turn, Pi release appears to drive structural changes that lead to
subunit dissociation. The reaction is inhibited by vanadate, an analog of Pi, indicating
that Pi release, and the rearrangements coupled to it, are essential for subunit dissocia-
tion (Savelsbergh et al. 2009 ) (Fig. 7.4c ). After the dissociation of the 50S subunit,
deacylated tRNA as well as mRNA remain bound to the 30S subunit and dissociate
rather slowly (Karimi et al. 1999 ; Peske et al. 2005 ). IF3 stimulates tRNA dissocia-
tion from the 30S subunit, followed by the release of mRNA, which renders the ribo-
some free for the next round of translation (Karimi et al. 1999 ; Peske et al. 2005 ) .

7.6.2.2 Distinct Functions of EF-G in tRNA Translocation

and Ribosome Recycling

Several aspects of EF-G action are similar in translocation and ribosome disassem-
bly. In both reactions, EF-G hydrolyzes GTP rapidly (Rodnina et al. 1997 ;