Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (ϳ180

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... amino acids) in the bacterialtype ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus. This non-cognate amino acid was efficiently acylated in vitro onto tRNA Pro , and the misacylated Cys-tRNA Pro was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases. Aminoacyl-tRNA synthetases (AARSs) 1 ensure accuracy in the translation of the genetic code by precisely selecting and attaching their cognate amino acids to the corresponding tRNA species (1). This is accomplished in a two-step process of amino acid activation and aminoacyl-tRNA formation. First, the enzyme-bound aminoacyl-adenylate is formed in the presence of