Figure 2.

Steady-state level of alanyl-tRNA^Ala. Total low molecular weight RNA was isolated from knockout cells producing the indicated form of tRNA^Ala under acidic conditions to preserve aminoayl-tRNA. (A) Direct identification of aminoacyl-tRNA and deacyl-tRNA by fractionation on an acidic polyacrylamide gel and detected by Northern blot analysis. HO⁻ indicates deacylation by Tris base. The arrowhead indicates alanyl-tRNA^Ala. The percent aminoacyl-tRNA^Ala (100 × aminoacyl-tRNA^Ala/{aminoacyl-tRNA^Ala + deacyl-tRNA^Ala}) shown is the average and standard error of three determinations, except the G⋅C sample is based on one measurement. (B) Direct identification of deacyl-tRNA^Ala by 3′ nucleoside resection (giving N-1 length chains) and resolution from full-length (N-length chains) tRNA^Ala on a DNA sequencing gel. IO₄⁻/β indicates treatment with periodate and β elimination. The arrowhead indicates N-1 tRNA^Ala. The percent deacyl-tRNA^Ala [100 × (N-1/{N-1 + N})] shown is the average and standard error of four determinations (two for G⋅C).