Almost all Escherichia coli tRNAs which have codons with a 5'-adenosine have an unusual nucleoside adjacent to the 3' end of the anticodon the systematic name of which is N -[N-(9-@-D-ribofuranosylpurin-6-yl)carbamoyllthreonine (@A). An enzyme has been isolated from E. coli which can synthesize this nucleoside in t R N A when provided with a t R N A deficient in t6A and with L-threonine, bicarbonate, ATP, and Mg2+. The t6A-deficient tRNA was isolated from a threonine-starved culture of an E. coli strain which is a threonine auxotroph and is also a relaxed control mutant (capable of synthesizing R N A in the absence of protein synthesis). The t6A-deficient t R N A was treated with periodate to destroy the 3'-ribose so that the enzyme assay could depend upon the incorporation of labeled L-threonine into the t R N A to form t6A without interference by the threonine incorporating activity of the E . coli threonyl-tRNA synthetase. Using this assay, an en-

No abstract