Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin

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Abstract
We have examined the effects of novobiocin and template topology on the transcription of two chloroplast genes encoding the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the chloroplast ATPase (atpB), in an in vitro transcription system. The template topology was monitored by agarose gel electrophoresis while the in vitro transcripts were determined by 5′ S1 nuclease analysis under identical conditions. We discovered that our chloroplast transcription extracts contain a DNA gyrase activity and a chromatographically separable topoisomerase I activity. Incubation of a supercoiled template with the extracts under the same conditions in which transcription assays were carried out leads to a decrease in the supercoiled from and concomitant appearance of distinct topoisomers. More extensive relaxation of the supercoiled template occurs when nucleotide triphosphates are omitted from the reaction mixture or when a low concentration (25 μg/ml) of novobiocin is added. Higher concentrations (≥ 250 μg/ml) of the drug, however, also inhibit the topoisomerase I activity. The transcription of the atpB gene is inhibited by lower concentrations of novobiocin as compared to the rbcL gene in the same reaction mixture. Relaxed, closed circular template and linearized DNA are not substrates for chloroplast transcription extracts, although they are transcribed accurately by the E. coli RNA polymerase under our conditions. Control of in vitro transcription of the two chloroplast genes by template topology can also be demonstrated by modulating the relative activity for the topoisomerases in the transcription extract. Our results suggest that changes in template topology may be a mechanism by which chloroplast genes are differentially regulated and the chloroplast DNA gyrase and topoisomerase I are key enzymes for this mode of regulation in vivo.