title: Further in vitro exploration fails to support the allosteric three-site model.
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.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Alexandros D
authors.initials: AD
authors.lastname: Petropoulos
authors.forename: Rachel
authors.initials: R
authors.lastname: Green
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D020029:Base Pairing; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D004926:Escherichia coli; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000302:isolation & purification; D012270:Ribosomes / Q000737:chemistry*
chemical_list: D003062:Codon; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl