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

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.

Evidence for Transcriptional Regulation of Plastid Photosynthesis Genes in Arabidopsis thaliana Roots

“…Influence of DNA superhelicity on transcriptional activities of plastid genes has also been proposed (Stirdivant et al, 1985;Lam and Chua, 1987;Thompson and Mosig, 1987). To investigate the altered activity of DNA templates in A. thaliana, we have performed the in vitro transcription with E. coli RNA polymerase.…”

“…Gene expression in plastids has been proposed to be regulated at multiple levels such as by DNA copy number (Aguettaz et al, 1987), DNA superhelicity (Stirdivant et al, 1985;Lam and Chua, 1987;Thompson and Mosig, 1987), DNA methylation (Ngernprasirtsiri et al, 1988;Kobayashi et al, 1990), and DNA transcription (Schrubar et al, 1990), and RNA stability (Deng The research was supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (Monbusho), and by grants from the New Energy and Industrial Technology Development Organization (NEDO)/ the Research Institute of Innovative Technology for the Earth (RITE), the Toray Science Foundation, and the Salt Science Research Foundation.…”

Regulation of Light-Induced Chloroplast Transcription and Translation in Eight-Day-Old Dark-Grown Barley Seedlings

“…In the present case, the transcriptional activity of plastid-encoded genes could be mediated by cytoplasmic protein(s) or intermediary regulatory molecules. Light-induced cytoplasmic protein synthesis could mediate plastid transcription by changes in the protein composition of the RNA polymerase ( 19), synthesis of sequence-specific DNA-binding factors ( 17), light-induced changes in local template topology (15) or the light-induced synthesis of prokaryotic-like sigma factors ( 19 4) and from illuminated seedlings incubated without (lanes 2 and 5, control) or with Tagetin (lanes 3 and 6) were fractionated, electrophoresed, and autoradiographed as described in Figure 4. Legends same as Figure 4 (2,9,26).…”

DNA Gyrase Is Involved in Chloroplast Nucleoid Partitioning

“…First, the inhibitors of bacterial DNA gyrase novobiocin and nalidixic acid inhibit chloroplast DNA synthesis in higher plants (Heinhorst et al, 1985;Mills et al, 1989). The same drugs also affect the accumulation of specific chloroplast transcripts in higher plants (Lam and Chua, 1987). Moreover, Arabidopsis thaliana genome analyses have predicted that the Arabidopsis DNA gyrase A and B subunits are targeted to organelles (Elo et al, 2003).…”