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. 2015 Oct 12;43(20):9905–9917. doi: 10.1093/nar/gkv1020

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© The Author(s) 2015. 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 1. — Functional characterization of T1 and T2. (A) Complementation assay of the E. coli conditional mutant IBPC6881(pSGUB4). Derived strains containing the empty vector pTrc99A (sector 1) or derived plasmids expressing the E. coli ThrRS (sector 2), T1 (sector 3), T1 and Anabaena tRNA^Thr(CGU) (sector 4), T2 (sector 5), T2 and Anabaena tRNA^Thr(CGU) (sector 6) were streaked on the indicated media. (B) Lack of complementation is not due to a deficient expression of T1 in E. coli. Western blot of extracts were carried out using 10 μg protein extracts from the strains used in the complementation assay that express the indicated products, cultured (+) or not (−) with 1 mM IPTG and incubating with anti-hexahistidine antibodies. Numbers indicate the corresponding sector for each strain in the complementation assay. (C) Aminoacylation assay with recombinant T1 or T2 proteins. Assays were carried out in a volume of 22 μl and contained 50 nmol of T1 or T2 and 5 μM Anabaena tRNA^Thr(CGU). Average values ± s.d. of three independent experiments are represented. (D) Northern blot with RNA from Anabaena cells subjected or not to incubation in the presence of chelating agent TPEN for the indicated period of time (in hours). Genes used as probes are indicated. (Northern assays of thrS2 in these conditions have been published elsewhere (38), and it is only shown here for the sake of comparison). (E) Quantitative Real-Time PCR analysis of transcripts of thrS1 and thrS2 from cells incubated or not with 20 μM TPEN for the time indicated in hours in the horizontal axis. Levels of the thrS1 gene in cells not incubated with TPEN (t = 0) were assigned the arbitrary value of 1 and all other measures were expressed in proportion to this value. Empty and solid bars correspond to thrS1 and thrS2, respectively. Average values ± SE of three independent biological replicas are represented. (F) Total tRNA (2 μg) from WT Anabaena cells or the deletion mutant MN42 (ΔthrS2) cultured for the indicated time (in hours) in the presence of 20 μM TPEN were resolved in denaturing urea-acrylamide acid gels, transferred to nylon membranes and hybridized with the indicated probes. Positions of aminoacylated tRNA and deacylated tRNA are labeled ‘a’ and ‘d’, respectively. (G) Serial dilutions of cell suspensions of WT Anabaena, the deletion mutant MN42 (ΔthrS2) or the insertion mutant MN8 (thrS2::C.S3) were spotted onto BG-11 solid medium supplemented or not with 10 μM TPEN as indicated. All experiments were repeated at least three times with identical results.