Expression of the Chlamydomonas reinhardtii chloroplast tRNA Glu gene in a homologous in vitro transcription system is independent of upstream promoter elements

Abstract

Chloroplast tRNA Glu is a bifunctional molecule involved in both the early steps of chlorophyll synthesis and chloroplast protein biosynthesis. Recently the enzymes involved in these processes have been characterized from the green alga Chlamydomonas reinhardtii. In order to investigate whether transcription of the gene for the tRNA Glu cofactor would be a possible point of regulation for the biosynthesis of chlorophyll, a homologous in vitro transcription system for C. reinhardtii chloroplast RNA polymerase was developed. The enzymatic activity was partially purified by ion-exchange chromatography to separate it from nuclear RNA polymerases. The highest rate of synthesis was found at pH 7.9, 40 m m KCl, 9 m m MgCl 2 and with 25 μg plasmid DNA containing the chloroplast tRNA gene per milliliter. The activity was not sensitive to high amounts of α-amanitin (500 μg/ml) and rifampicin, but was clearly inhibited by heparin. This system was used to undertake a promoter analysis of one of the two identical tRNA Glu gene copies found in the C. reinhardtii chloroplast genome ( trnE1). The analyzed tRNA gene behaved like a single transcription unit driven by its own promoter. The transcript terminated in a run of four consecutive T residues downstream of the gene. The nucleotide sequence in the 5′ region of the gene revealed several potential promoter elements with homology to known chloroplast promoters of the “−10 and −35 region” and the “ Euglena promoter” types. Surprisingly, deletion of the complete 5′ region did not affect in vitro transcription, while partial deletions of the 5′ and 3′ coding region totally abolished transcription. This indicates the presence of an internal control region previously found for genes transcribed by nuclear RNA polymerase III. Protein binding studies with the coding region of trnE1 using gel retardation assays demonstrated the formation of two differently sized complexes. In vitro transcription of the tRNA Glu gene in extracts prepared from light and dark grown algae failed to demonstrate any significant influence of light on the transcription reaction.