Journal: Nucleic Acids Research

Article Title: Life without tRNA Ile -lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA 2 Ile

doi: 10.1093/nar/gkt1009

Figure Lengend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

Article Snippet: For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

Techniques: Purification, Mutagenesis, Labeling, Control, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

Journal: Nucleic Acids Research

Article Title: Life without tRNA Ile -lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA 2 Ile

doi: 10.1093/nar/gkt1009

Figure Lengend Snippet: RNA sequencing of the anticodon loop of wild-type tRNA 1 and mutant tRNA 1 by MS analysis of RNase T1 fragments

Article Snippet: For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

Techniques: RNA Sequencing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Life without tRNA Ile -lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA 2 Ile

doi: 10.1093/nar/gkt1009

Figure Lengend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

Article Snippet: For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

Techniques: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay