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. 2020 Feb 4;48(6):3071–3088. doi: 10.1093/nar/gkaa055

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — The proportion of charged tRNAs is not reduced by tRNA synthetase depletion. (A) Northern blots of control tRNA^Arg_UCU and tet-off tRNA^Lys_CUU isolated from a tetO-KRS1 (lysyl -tRNA synthetase shut-off) strain grown in a range of doxycycline concentrations up to 0.5 μg ml⁻¹. Blots include a control lane of pH 8-treated tRNA to provide a marker of deacylated tRNA (open arrow, ‘deacl’). The slower migration position of acylated tRNA is indicated (closed arrowhead). Results shown are typical of other biologically independent experimental replicates. There is a linear relationship between signal and image intensity. The images shown are of single blots and are not composite (2μg total RNAs loaded per lane). (B) Quantification of the northern blot showing average % tRNA charging (UCU-Arg; closed circles) and CUU-Lys (closed squares). Means of three biological replicates on independent blots ± standard deviation (n = 3). (C) Northern blots of control tRNA^Lys_CUU and tet-off tRNA^Gln_UUG isolated from a tetO-GLN4 (glutaminyl-tRNA synthetase shut-off) strain grown in a range of doxycycline concentrations, and band intensities quantified (D; UUG-Gln; closed circles and CUU-Lys; closed squares; 2 μg total RNAs loaded per lane).