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J. Biol. Chem., Vol. 278, Issue 17, 15095-15104, April 25, 2003
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From the Department of Biochemistry and Centre for Gene Research,
University of Otago, P. O. Box 56, Dunedin, New Zealand and
§ Max-Planck-Institut für Molekulare Genetik,
Ihnestrasse 73, D-14195 Berlin, Germany
The function of the decoding release
factor (RF) in translation termination is to couple cognate recognition
of the stop codon in the mRNA with hydrolysis of the completed
polypeptide from its covalently linked tRNA. For this to occur, the RF
must interact with specific A-site components of the active centers
within both the small and large ribosomal subunits. In this work, we
have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor
RF2, during translation termination. In the presence of the cognate UGA
stop codon, residues flanking the universally conserved
250GGQ252 motif of RF2 were each shown to
footprint to the large ribosomal subunit, specifically to conserved
elements of the peptidyltransferase and GTPase-associated centers.
In contrast, residues that flank the putative "peptide anticodon"
of RF2, 205SPF207, were shown to make a
footprint in the small ribosomal subunit at positions within well
characterized 16 S rRNA motifs in the vicinity of the decoding center.
Within the recently solved crystal structure of E. coli
RF2, the GGQ and SPF motifs are separated by 23 Å only, a distance
that is incompatible with the observed cleavage sites that are up to
100 Å apart. Our data suggest that RF2 may undergo gross
conformational changes upon ribosome binding, the implications of which
are discussed in terms of the mechanism of RF-mediated termination.
Mapping Functionally Important Motifs SPF and GGQ of the Decoding
Release Factor RF2 to the Escherichia coli Ribosome by
Hydroxyl Radical Footprinting
IMPLICATIONS FOR MACROMOLECULAR MIMICRY AND STRUCTURAL CHANGES
IN RF2*
,
*
This work was supported by Postgraduate Scholarships from
the University of Otago and the Health Research Council of New Zealand (to D.-J. G. S.), the Royal Society of New Zealand Marsden Fund (to
W. P. T.), at the beginning an International Research Scholar Award
from the Howard Hughes Medical Institute (to W. P. T.), and a Human
Frontier Science Program Grant Number RG32/97 (to Y. Nakamura (Tokyo),
L. Kisselev (Moscow), M. Philippe (Paris), and W. P. Tate
(Dunedin)).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Malaghan Institute of Medical Research, P.O. Box
7060, Newtown, Wellington South, New Zealand.
¶
Supported by the Alexander von Humboldt Foundation.
To whom correspondence should be addressed. Tel.:
64-3-479-7864; Fax: 64-3-479-7866; E-mail:
warren.tate@stonebow.otago.ac.nz.
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