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Selective in vitro transcription of chloroplast genes

Transcription of Euglena gracilis chloroplast genes has been investigated by using in vitro transcription systems. A DNA‐dependent RNA polymerase responsible for the transcription of rRNA genes has been isolated as a nucleoprotein complex (transcriptionally active chromosome). The RNA polymerase remains tightly bound to the chloroplast DNA template and does not initiate transcription with cloned chloroplast genes. A transcriptionally active extract has been prepared from intact Euglena chloroplasts. The soluble RNA polymerase in this extract recognizes cloned chloroplast tRNA genes and tRNA‐sized products have been detected after transcription. The tRNA‐sized molecules specifically hybridize to the tRNA genes in the plasmid DNA. At least five tRNA‐sized products have been identified from transcription of a trnY1‐trnH1‐trnM1‐trnE1‐trnW1‐trnG1 cluster. Evidence is also presented that processing enzymes in the chloroplast‐extract can recognize a polycistronic tRNAVal‐tRNAAsn‐tRNAArg precursor and process it into tRNA‐sized molecules. Truncated templates have been used to demonstrate that the chloroplast tRNA genes are actively transcribed. From a comparison of 5′ flanking sequences in chloroplast tRNA genes, a consensus sequence which might function as a promoter, has been identified. The properties of the RNA polymerase involved in the transcription of chloroplast rRNA genes and tRNA genes have been investigated and compared.