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R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria Escherichia coli
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.ORCID iD: 0000-0002-7124-792X
2017 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 36, p. 15134-15142Article in journal (Refereed) Published
Abstract [en]

The current understanding of the specificity of the bacterial class I release factors (RFs) in decoding stop codons has evolved beyond a simple tripeptide anticodon model. A recent molecular dynamics study for deciphering the principles for specific stop codon recognition by RFs identified Arg-213 as a crucial residue on Escherichia coli RF2 for discriminating guanine in the third position (G3). Interestingly, Arg-213 is highly conserved in RF2 and substituted by Ile-196 in the corresponding position in RF1. Another similar pair is Leu-126 in RF1 and Asp-143 in RF2, which are also conserved within their respective groups. With the hypothesis that replacement of Arg-213 and Asp-143 with the corresponding RF1 residues will reduce G3 discrimination by RF2, we swapped these residues between E. coli RF1 and RF2 by site-directed mutagenesis and characterized their preference for different codons using a competitive peptide release assay. Among these, the R213I mutant of RF2 showed 5-fold improved reading of the RF1-specific UAG codon relative to UAA, the universal stop codon, compared with the wild type (WT). In-depth fast kinetic studies revealed that the gain in UAG reading by RF2 R213I is associated with a reduced efficiency of termination on the cognate UAA codon. Our work highlights the notion that stop codon recognition involves complex interactions with multiple residues beyond the PXT/SPF motifs. We propose that the R213I mutation in RF2 brings us one step forward toward engineering an omnipotent RF in bacteria, capable of reading all three stop codons.

Place, publisher, year, edition, pages
2017. Vol. 292, no 36, p. 15134-15142
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-335202DOI: 10.1074/jbc.M117.785238ISI: 000409902100035PubMedID: 28743745OAI: oai:DiVA.org:uu-335202DiVA, id: diva2:1163143
Funder
Swedish Research Council, 2016-06264, 2013-8778, 2014-4423, 2008-6593Knut and Alice Wallenberg Foundation, KAW2011.0081Available from: 2017-12-06 Created: 2017-12-06 Last updated: 2018-01-13Bibliographically approved

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Korkmaz, GürkanSanyal, Suparna

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