Transcription of E. coli and Euglena chloroplast tRNA gene clusters and processing of polycistronic transcripts in a HeLa cell-free system.

The transcription of cloned tRNA genes that are clustered in the Escherichia coli and Euglena gracilis chloroplast genome was studied in a HeLa cell-free extract. RNA polymerase III transcribes the tRNA gene clusters into polycistronic primary transcripts, consisting of pre-tRNAVal-tRNAAsn-tRNAArg and pre-tRNAAsp-tRNATrp transcribed from the Euglena chloroplast and E. coli tRNA locus, respectively. A UV5-lac promoter in the 5'-flanking sequence of the E. coli tRNA gene cluster can be removed without effect on transcription initiation frequency. During transcription of the tRNA gene clusters, RNA polymerase III initiates predominantly, if not solely, with the first tRNA gene. Upon removal of the first tRNA gene, RNA polymerase III initiates transcription with the following tRNA gene. The primary transcripts are subsequently processed to mature tRNAs. RNA fingerprint analysis and reincubation experiments were used to establish the steps of tRNA maturation of the pre-tRNAVal-tRNAAsn-tRNAArg. The 3' trailer is processed prior to the removal of 5' leader and before cleavage of the primary transcript in the spacer sequences. The pre-tRNAs are then processed to mature tRNA molecules.