Accurate processing and pseudouridylation of chloroplast transfer RNA in a chloroplast transcription system

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Abstract
The trancription of a cloned trnV1-trnN1-trnR1 cluster from Euglena gracilis chloroplast (ct) DNA and the processing of a tRNAVal-tRNAAsn-tRNAArg polycistronic precursor were studied in a spinach ct transcription extract. A soluble ct RNA polymerase selectively transcribes the trnV1-trnN1-trnR1-trnL1 locus in the EcoG fragment from the Euglena ct genome. Restriction enzyme modified templates and RNA fingerprint analysis were used to confirm that the tRNA genes were correctly transcribed. The tRNAVal-tRNAAsn-tRNAArg polycistronic precursor transcribed by RNA polymerase III in a HeLa cell extract was used as a substrate to demonstrate that a ct tRNA precursor molecule is correctly processed by the ct tRNA processing enzymes. The oligonucleotide pattern of tRNAs processed in vitro from the tRNAVal-tRNAAsn-RNAArg polycistronic precursor is indistinguishable from tRNAVal, tRNAAsn and tRNAArg transcribed by the ct RNA polymerase and processed in the ct transcription extract. The 3′-CCAOH is added to the tRNAs by a 3′ nucleotidyltransferase after correct processing of the 3′ terminus. Correct pseudouridylation was demonstrated for uridine residues in a tRNAMet m molecule transcribed from a spinach ct trnM1 locus. Thus, the enzymatic activities involved in tRNA biosynthesis in vitro include DNA-dependent (tDNA) RNA polymerase, a 5′-processing activity (RNase P-like), a 3′-exonuclease, an endoribonuclease involved in 3′-tRNA maturation, a tRNA nucleotidyltransferase, and pseudouridylate synthetase.