Abstract
The regulation of gene expression is still one of the major issues in modern plant molecular biology. The amount of RNA in a cell is regulated by both transcriptional and posttranscriptional events. Methods to determine these steady-state levels of RNAs, such as Northern analysis, ribonuclease protection assay (RPA), and quantitative real-time PCR, do not discriminate between regulation by de novo RNA synthesis and the influence by degradation or stabilization. To assess the rate of transcription of individual genes, run-on transcription is utilized. To this end, isolated chloroplasts are used in brief in vitro transcription reactions in the presence of radiolabeled nucleotides, with a subsequent hybridization of the isolated RNA with DNA fragments spotted on membranes. Here, we describe a protocol for run-on transcription in chloroplasts isolated from Arabidopsis leaves and present data on the transcriptional activity of several plastid genes in detached leaves of different Arabidopsis ecotypes.
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Acknowledgments
This work was funded by Deutsche Forschungsgemeinschaft (SFB 429 to T.B. and K/L.). We thank Liliana Borsellino, Dr. Victor V. Kusnetsov, and Dr. Maria V. Yamburenko for their helpful discussions.
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Zubo, Y.O., Börner, T., Liere, K. (2011). Measurement of Transcription Rates in Arabidopsis Chloroplasts. In: Jarvis, R. (eds) Chloroplast Research in Arabidopsis. Methods in Molecular Biology, vol 774. Humana Press. https://doi.org/10.1007/978-1-61779-234-2_11
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DOI: https://doi.org/10.1007/978-1-61779-234-2_11
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