T4 RNA ligase will catalyze the addition of nucleoside 3′,5′-bisphos-phates Onto the 3′ terminus of tRNA resulting in a tRNA molecule one nucleotide longer with a 3′ terminal phosphate. Under appropriate conditions the reaction is quantitative and, if high specific radioactivity bisphosphates are used, it provides an efficient means for in vitro labeling of tRNA. Although the 3′ terminal hydroxyl is a good acceptor, the 5′ terminal phosphate in most tRNA is not an effective donor in the RNA ligase reaction. This poor reactivity is due to the secondary structure of the 5′ terminal nucleotide. If E. coli tRNAfMet is used, the 5′ phosphate is reactive and the major product with RNA ligase is the cyclic tRNA.
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