BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 380–385 (1997) ARTICLE NO. RC976464 Transcription and Processing of the Gene for Spinach Chloroplast Threonine tRNA in a Homologous in Vitro System Yi-Sheng Cheng, Chi-Hui Lin, and Liang-Jwu Chen Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan, ROC Received February 26, 1997 mapped between trnE and the psbD operon in the large An in vitro system was established to study the tran- single copy region, has been shown to be a monocis- scription and processing of threonine tRNA using tronic tRNA gene (11, 12). Since no further promoter spinach chloroplast enzyme extract. Experiments us- study for the trnT gene has been reported and no pro- ing a series of 5 deletion mutants demonstrated that karyote-like promoter sequences were found 5 of this the transcription of trnT gene required no 5 upstream gene, we speculated that this trnT gene might be an- promoter elements. Four plasmid DNA templates con- other chloroplast gene, in addition to the trnR, trnS and taining trnT were constructed for tRNA processing trnE genes (3, 13), that requires no upstream promoter assay. The processing reaction was carried out either elements for transcription. with exogenously added precursor-tRNAs made by T7 In chloroplasts, tRNAs are processed immediately RNA polymerase or with RNAs synthesized by the after transcription. So, it is difficult to monitor the in transcription activity in the same processing enzyme vivo transcription and processing activities separately. extract. Both assays demonstrated that the 5 and 3  Previous in vitro studies on transcription activity and ends of mature tRNA were processed endonucleolyti- cally and the processing of the 5 end preceded the promoter structure of tRNA genes were based on the maturation of the 3  end. The activity of nucleotidyl production of the mature tRNAs (8). Since the chloro- transferase that adds CCA nucleotides to the 3  end of plast enzyme extract prepared for in vitro assay usually tRNA was also observed. The use of a coupled tran- contains both transcription and processing activities, scription and processing system provides us with a the tRNA thus produced is a result of both activities better insight to the tRNA processing mechanism of (2, 14). With no detectable leader and trailer segments, the chloroplast.  1997 Academic Press the processing activities involved could not be charac- terized. Therefore, precursor tRNAs synthesized in vitro have to be used as substrates in order to study the pertinent processing activities. To date, many chlo- All the tRNAs involved in chloroplast protein synthesis roplast tRNA processing activities have been charac- are believed to be encoded in the chloroplast genome (1). terized by this approach (5-7, 15, 16), but no coupled In higher plants, chloroplast tRNAs are usually tran- transcription/processing activities have been well char- scribed as monocistronic precursors from their own pro- acterized. moters (2-4) and processed post-transcriptionally (2, 5-7) In this study, the spinach chloroplast enzyme ex- to form the mature tRNAs. Two types of promoters for tract was used to examine both transcription and pro- chloroplast tRNA genes have been reported. One contains cessing activities against the spinach chloroplast the prokaryote-like upstream ‘‘010’’ and ‘‘035’’ consensus threonine tRNA. Using this system, we were able to sequences such as the spinach trnM2 (8), and the other, observe the processed intermediates in addition to such as the spinach trnR and trnS, requires no upstream the mature tRNA during the tRNA maturation pro- promoter elements (3). The primary transcript produced cesses. Results in this study demonstrated that the by either type of promoter still requires a series of pro- spinach chloroplast trnT gene requires no upstream cessing events including 5 end and 3  end cleavages, promoter elements for transcription, and the matura- CCA addition at the 3  end, and some base modifications tion process involves a 5 cut prior to the 3  cut of its to form the mature tRNA molecules. precursor. The CCA addition to the 3  end of tRNA The spinach chloroplast trnT-GGU has been se- quenced and characterized (9, 10). Its gene, which was was also demonstrated. 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 380