Figure 4.

Import signal and identity elements for mitochondrial GlnRS are distinct. (A) Inferred secondary strucures of cytosol-specific tRNA^Gln (Cyt) and mutant tRNA^Gln(D-Ile), which is imported into mitochondria (Cyt/Mit) (20). Nucleotides in the tRNA^Gln(D-Ile) that are different from the cytosolic ones are boxed. (B) Cytosol-specific tRNA^Gln (CUG) and mutant imported tRNA^Gln [CUG(D-Ile)] were charged with ¹⁴C-glutamine using 60 μg each of RNA-depleted cytosolic (Cyt) or mitochondrial matrix (Mit) fractions. As a control, the reactions were performed without adding tRNAs (no tRNA). Y axis as Fig. 1. (C) Competition assays were performed by charging of mitochondrial tRNA^Gln (UUG) with ¹⁴C-glutamine using 60 μg of mitochondrial matrix (Mit) in the presence of a 1- or 5-fold excess of cytosolic tRNA^Gln (CUG) (Left) or the mutant-imported tRNA^Gln [CUG(D-Ile)] (Right). The level of charging is expressed as percentage of a standard charging reaction without competitor tRNA. Bars indicate mean values (±SD) of independent aminoacylation reactions (n = 3–8) using at least two independently prepared cytosolic or mitochondrial extracts.