PMID: 8600459Mar 1, 1996Paper

In vivo deuteration of transfer RNAs: overexpression and large-scale purification of deuterated specific tRNAs

Nucleic Acids Research
R JünemannK H Nierhaus

Abstract

Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.

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Citations

Jun 28, 2000·Nucleic Acids Research·E CayamaF J Triana-Alonso
Aug 12, 2014·Science·Matthieu G GagnonThomas A Steitz
Sep 10, 2009·Chembiochem : a European Journal of Chemical Biology·Keitaro EbisuMasumi Taki
Oct 14, 2014·Molecular Cell·Yury S PolikanovPetr V Sergiev
May 20, 2015·Nature Structural & Molecular Biology·Raktim N RoyThomas A Steitz
Apr 20, 2016·Proceedings of the National Academy of Sciences of the United States of America·Matthieu G GagnonThomas A Steitz
Sep 2, 2004·The Journal of Biological Chemistry·Fabien PierrelMohamed Atta
Aug 8, 2018·Acta Crystallographica. Section D, Structural Biology·Emilie Mahieu, Frank Gabel
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Nov 15, 2016·RNA Biology·Pius SchrodeMatthias Erlacher
Feb 16, 2019·Science·Christopher D RaeV Ramakrishnan
Aug 19, 2014·Nature Structural & Molecular Biology·Yury S PolikanovC Axel Innis
Dec 25, 2019·Nature Structural & Molecular Biology·Dejian ZhouMatthieu G Gagnon
Aug 21, 2020·Science·Michael William WebsterAlbert Weixlbaumer
Jun 13, 2021·Communications Biology·Tolou GolkarAlbert M Berghuis

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