Synthesis and maturation of Xenopus laevis methionine tRNA gene transcripts in homologous cell-free extracts

Abstract

A cloned initiator methionine tRNA gene from Xenopus laevis has been transcribed in cell-free extracts (S-100) prepared from cultured X. laevis kidney cells. RNA polymerase III produces two primary transcripts of this gene which initiate with a pppG and a pppA located seven and four nucleotides, respectively, in front of the mature tRNAMet1 5' end. Both terminate with a dT5 tract just beyond the nucleotides encoding the mature tRNA. The tRNA precursors are readily processed in vitro to mature length tRNA which contains six of the seven modified nucleotides found in the in vivo tRNAMet1. Many of these ribonucleotide modifications (m1G, m2G, m7G, D, and m1A) are introduced into the primary transcripts. The single exception is t6A which is found in the tRNAMet1 anticodon loop only after maturation of the 5' and 3' termini.