11] Enzymic modification of the C-C-A terminus of tRNA

Abstract

Publisher Summary The chapter discusses the enzymic modification of the C-C-A terminus of tRNA. The tRNA species with an altered C-C-A terminus are used for investigation of the mechanism of aminoacylation and ribosomal protein biosynthesis, as well as for spectroscopic and X-ray crystallographic studies. ATP(CTP):tRNA nucleotidyltransferase (EC 2.7.7.25), which catalyzes the incorporation of CMP and AMP into tRNA lacking the C-C-A part of its 3' terminus, is used for preparation of modified tRNA species as it has been shown that some analogs of ATP and CTP are also substrates for this enzyme. An essential prerequisite for the preparation of uniformly modified tRNAs via incorporation of AMP and CMP analogs by ATP(CTP) : tRNA nucleotidyltransferase into the 3' end of tRNA is a tRNA species with a uniformly shortened 3' terminus. Furthermore, a highly purified enzyme free of nuclease is required because long incubation times and high enzyme concentrations usually have to be applied owing to the higher Km values and lower reaction velocities with the ATP and CTP analogs. Procedures for the isolation of ATP(CTP), tRNA nucleotidyltransferase from yeast, preparation of tRNAPhe-A, tRNAPhe-A-C, and tRNAPhe-A-C-C from yeast, enzymic synthesis of modified tRNAs, and their analysis are described.