Methods for Studying the Interactions of Translation Factors with the Ribosome - Biophysical Approaches to Translational Control of Gene Expression - page 86

radicals while converting Fe(II) into Fe(III). The role of ascorbate is to regenerate
Fe(II)-EDTA by reducing Fe(III) back to Fe(II), and allow continuous hydroxyl
radical production (reviewed in Ehresmann et al. 1987 ; Kolupaeva et al. 2007 ) .

5.4.2 Applications

Before high-resolution structures became available, footprinting studies were instru-
mental in mapping contact interfaces between the two ribosomal subunits and bind-
ing sites for tRNAs, translation factors, and antibiotics (reviewed in Fraser and
Doudna 2007 ). When the crystal structures of the ribosome were reported, com-
parative analysis of the available footprinting data with the actual positions of ribo-
somal proteins and RNAs confirmed that footprinting can provide reliable
information about the overall position of proteins on the ribosome (Whirl-Carrillo
et al. 2002 ). More recently, Pestova and co-workers showed that the helicase DHX29
is required for scanning through stable stems in the 5¢-untranslated region (5¢ -UTR)
of mRNA. Chemical and enzymatic footprinting indicated that DHX29 likely binds
to 18S rRNA h16 near the mRNA Entry channel of the small ribosomal subunit, in
line with its function (Pisareva et al. 2008 ) .
Another application of footprinting that has become popular in recent years is
tracking conformational rearrangements in ribosomal complexes at different stages
of translation or upon binding of antibiotics using changes in footprinting patterns.
An important feature of the approach is that when the structure of the ribosome at
specific stages is known from X-ray crystallography or Cryo-EM, the observed
footprinting patterns can be readily correlated to specific ribosomal conformations.
For example, Noller and co-workers used chemical footprinting, in combination
with other assays, to identify the effects of EF-G in complex with GTP, GDPNP (a
non-hydrolyzable GTP analog), and GDP on the positions of mRNA and tRNAs in
translocation. They found that GTP is required for complete translocation to occur
and for the release of Exit site (E-site) tRNA, whereas EF-G in complex with
GDPNP or GDP fusidic acid only causes movement of the Aminoacyl-tRNA site

(A-site) and Peptidyl-tRNA site (P-site) tRNAs to hybrid A/P and P/E states, respec-
tively (Spiegel et al. 2007 ). Green and co-workers reported that stop codon recogni-
tion by release factors induced specific conformational changes in the decoding
center of the small ribosomal subunit, which may help in promoting peptide release
(Youngman et al. 2007 ). A combination of footprinting and FRET was used to elu-
cidate the mechanism of action of the antibiotic viomycin, which appears to lock the
ribosome in a translocation intermediate (Ermolenko et al. 2007 ) .
Time-resolved hydroxyl radical footprinting is a variant of the method that allows
studying the dynamics of ribosomal complexes. Synchrotron X-ray irradiation is
typically used to generate hydroxyl radicals, since the approach requires large
amounts of hydroxyl radicals to be produced over a short time period (reviewed in
Fraser and Doudna 2007 ) .

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