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J Biol Chem. 2012 Apr 6;287(15):11642-8. doi: 10.1074/jbc.C111.330068. Epub 2012 Feb 29.

Further in vitro exploration fails to support the allosteric three-site model.

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1
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Abstract

Ongoing debate in the ribosome field has focused on the role of bound E-site tRNA and the Shine-Dalgarno-anti-Shine-Dalgarno (SD-aSD) interaction on A-site tRNA interactions and the fidelity of tRNA selection. Here we use an in vitro reconstituted Escherichia coli translation system to explore the reported effects of E-site-bound tRNA and SD-aSD interactions on tRNA selection events and find no evidence for allosteric coupling. A large set of experiments exploring the role of the E-site tRNA in miscoding failed to recapitulate the observations of earlier studies (Di Giacco, V., Márquez, V., Qin, Y., Pech, M., Triana-Alonso, F. J., Wilson, D. N., and Nierhaus, K. H. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 10715-10720 and Geigenmüller, U., and Nierhaus, K. H. (1990) EMBO J. 9, 4527-4533); the frequency of miscoding was unaffected by the presence of E-site-bound cognate tRNA. Moreover, our data provide clear evidence that the reported effects of the SD-aSD interaction on fidelity can be attributed to the binding of ribosomes to an unanticipated site on the mRNA (in the absence of the SD sequence) that provides a cognate pairing codon leading naturally to incorporation of the purported "noncognate" amino acid.

PMID:
22378789
PMCID:
PMC3320913
DOI:
10.1074/jbc.C111.330068
[Indexed for MEDLINE]
Free PMC Article
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