Biomedical Engineering Reference
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targets remains obscure, the major classes affect elongation events including decoding,
peptide bond formation, and/or translocation (Blanchard et al.
2010
; Carter et al.
2000
; Schlunzen et al.
2001
; Pioletti et al.
2001
). A major problem for national health
is multi-drug resistant bacteria. Mechanistic studies of the ribosome will help to find
new combination therapies that can be used to combat resistant bacteria. Specifically,
molecular dynamics simulations shed new light on the conformational transitions
between states that occur during protein synthesis. A future class of antibiotics could
be those that target transitions, rather than the major states of the ribosome.
3.2
tRNA Dynamics
The tRNA itself shows large fluctuations during translation. It binds to the ribosome
in a highly kinked configuration called the A/T state where the “A” represents the
anticodon interacting with the 30S subunit A site and the “T” represents the ternary
complex interacting with the 50S subunit at the GTPase activity center (Fig.
3.5
)
(Valle et al.
2002,
2003a
). Single molecule FRET studies also show rapid tRNA
fluctuations between several intermediates occurring before accommodation, where
the tRNA moves into the fully bound “A/A” position (anticodon interacting with the
30S A site and 3¢-CCA end interacting with the 50S A site). These include transi-
tions between the initial binding configuration, the codon-anticodon recognition
configuration, the GTPase activation configuration, and the A/T state. This led to
the conclusion that the aminoacyl-tRNA samples a broad ensemble of configurations
prior to accommodation (Geggier et al.
2010
) .
In addition to tRNA selection, cryo-EM reconstructions of intermediates during
translocation demonstrate tRNA flexibility. After accommodation and peptidyl
transferase, the tRNA in the A/A position moves to the A/P position (anticodon in
30S A site and 3¢-CCA end in 50S P site), while the tRNA in the P/P position moves
to the P/E position (anticodon in the 30S P site and 3¢-CCA end in the 50S E site).
Cryo-EM reconstructions show the tRNA to be in a curved configuration, albeit a
curved configuration which differs from the curved configuration required for the
A/T position (Connell et al.
2008
) . These two curved con fi gurations are suggestive
of an energy storage-release mechanism that allows the tRNA to propel itself
through the ribosome (Frank et al.
2005
). This enthalpic energy source would of
course be combined with the source of gated thermal fluctuations resulting from the
thermal bath (Whitford et al.
2010a
) .
The motions of tRNA within the ribosome have been the subject of intense inves-
tigation using both bulk- and single-molecule techniques. smFRET studies measur-
ing time-dependent changes in the intermolecular distance between A- and P-site
tRNAs have proven particularly revealing (Munro et al.
2007
) . During tRNA selec-
tion aa-tRNA was shown to enter the A site through reversible excursions between
multiple intermediate states. By analyzing the nature of these dynamics for both
correct (cognate) and incorrect substates (near-cognate), these dynamics were shown
to be directly related to the fidelity mechanism. After peptide-bond formation, but
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