Significance RNA structures control many essential functions of life, including gene expression, protein synthesis, and development. To perform these functions, RNAs often adopt complex tertiary folds. Here, we study the thermodynamics of late-transcriptional folding of eukaryotic and prokaryotic tRNAs. In near-cellular conditions, tRNAs gain folding cooperativity only when nearly all nucleotides in the acceptor stem are transcribed. Furthermore, the native 5′ and 3′ extensions in the precursor do not affect the folding pathway. Therefore, tRNA folding cooperativity is controlled by a single nucleotide under near-cellular conditions.