Guanylation of transfer ribonucleic acid by a cell-free lysate of rabbit reticulocytes.

The guanylation of tRNA in a cell-free system has been achieved in a lysate of rabbit reticulocytes. The enzymatic nature of the reaction is indicated by sensitivity to trypsin, heat inactivation, and by the precipitation of the guanylating activity with ammonium sulfate. The lysate did not incorporate guanine after endogenous tRNA was removed by RNase, or by binding the tRNA to an anion exchange resin. Guanylating activity was restored only by adding back reticulocyte or yeast tRNA. Liver tRNA or the synthetic homopolymers poly(A), poly(U), poly(C), or poly(G) did not serve as substrates. The reaction requires a monovalent cation which is best met by Li+ or K+ and the K, for guanine is 2.5 X lop5 M. The guanylated tRNA appears to be identical with that produced in uivo since the guanine residue is incorporated into an internal position in the polynucleotide chain and the guanylated tRNA co-chromatographs with the minor reticulocyte tRNAHiS on a reversed phase column. When uniformly labeled guanosine is the substrate the purine ring but not the ribose moiety is incorporated into tRNA.