Affinity Labelling of tRNA Nucleotidyltransferase from Baker's Yeast with tRNAPhe Modified on the 3′‐Terminus

Abstract

2′‐Deoxy‐2′‐amino‐cytidylic acid can be incorporated into position 75 of tRNA^Phe from yeast by tRNA nucleotidyltransferase yielding tRNA^Phe‐C‐C(2′NH₂). tRNA^Phe‐C‐C(2′NH₂) can be reacted with the N‐hydroxysuccinimide esters of bromoacetic acid and of mercuriacetic acid to yield the derivatives tRNA^Phe‐C‐C(2′NHCOCH₂Br) and tRNA^Phe‐C‐C(2′NHCOCH₂Hg⁺OH^‐). Each of these reactive tRNAs inactivates tRNA nucleotidyltransferase from yeast with similar kinetics. The enzyme can be protected against inhibition by its substrates tRNA^Phe‐C and tRNA^Phe ‐C‐C as well as ATP and CTP. A covalent. isolatable 1:1 complex between tRNA^Phe ‐C‐C(2′NHCOCH₂Br) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p‐hydroxymerecuribenzoate.