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 tRNAPhe from yeast by tRNA nucleotidyltransferase yielding tRNAPhe-C-C(2'NH2). tRNAPhe-C-C(2'NH2) can be reacted with the N-hydroxysuccinimide esters of bromoacetic acid of mercuriacetic acid to yield the derivatives tRNAPhe-C-C(2'NHCOCH2Br) and tRNAPhe-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 tRNAPhe-C and tRNAPhe-C-C as well as ATP and CTP. A covalent, isolatable 1:1 complex between tRNAPhe-C-C(2'NHCOCH2Br) and the enzyme was formed, but could not be found when the enzyme had previously been inactivated with p-hydroxymercuribenzoate.