Biosynthesis of Chloroplast Transfer RNA in a Spinach Chloroplast Transcription System

We have developed a chloroplast in vitro transcription system capable of transcribing tRNA genes (trn) from the spinach and Euglena gracilis chloroplast genomes. The RNA polymerase contained in the chloroplast extract transcribes the spinach chloroplast trnM2, trnV1, and trnl1 loci and the trnV1-trnN1-trnR1-trnL1 cluster in the EcoG fragment of the Euglena chloroplast genome. Restriction enzyme modified templates were used to demonstrate that the tRNA genes are transcribed in vitro. RNA fingerprint analysis confirmed that tRNAMetm, tRNAlle1 and tRNALeu are correctly processed transcripts from the spinach chloroplast trnM2, trnl1, and Euglena trnL1 loci respectively. CCAOH is added to the mature tRNAs in vitro by a 3' nucleotidyl transferase present in the chloroplast extract. Deletion mutants were constructed from the trnM2 locus to evaluate the role of 5' flanking sequences in transcription initiation and processing. DNA sequences between positions -56 to -85 upstream of the trnM2 locus are required for maximal transcription of tRNAMetm, but are not essential for processing. The RNA polymerase involved in chloroplast trn transcription is distinguishable from the RNA polymerase isolated as a DNA-protein complex from spinach chloroplast that is active in rRNA transcription.