Transcription and processing of the gene for spinach chloroplast threonine tRNA in a homologous in vitro system

Abstract

An in vitro system was established to study the transcription and processing of threonine tRNA using spinach chloroplast enzyme extract. Experiments using a series of 5' deletion mutants demonstrated that the transcription of trnT gene required no 5' upstream promoter elements. Four plasmid DNA templates containing trnT were constructed for tRNA processing assay. The processing reaction was carried out either with exogenously added precursor-tRNAs made by T7 RNA polymerase or with RNAs synthesized by the transcription activity in the same processing enzyme extract. Both assays demonstrated that the 5' and 3' ends of mature tRNA were processed endonucleolytically and the processing of the 5' end preceded the maturation of the 3' end. The activity of nucleotidyl transferase that adds CCA nucleotides to the 3' end of tRNA was also observed. The use of a coupled transcription and processing system provides us with a better insight to the tRNA processing mechanism of the chloroplast.