Abstract
Transcription is a highly complex biological process, with extensive layers of regulation, some of which remain to be fully unveiled and understood. To be able to discern the particular contributions of the several transcription steps it is crucial to understand RNA polymerase dynamics and regulation throughout the transcription cycle. Here we describe a new nonradioactive run-on based method that maps elongating RNA polymerases along the genome. In contrast with alternative methodologies for the measurement of nascent transcription, the BioGRO method is designed to minimize technical noise that arises from two of the most common sources that affect this type of strategies: contamination with mature RNA and amplification-based technical biasing. The method is strand-specific, compatible with commercial microarrays, and has been successfully applied to both yeasts Saccharomyces cerevisiae and Candida albicans. BioGRO profiling provides powerful insights not only into the biogenesis and regulation of canonical gene transcription but also into the noncoding and antisense transcriptomes.
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Acknowledgments
The authors are grateful to Jesica Portero for her help with Affymetrix arrays, to Lola de Miguel and Sebastián Chávez from the Universidad de Sevilla for providing RNA pol II ChIP -chip data, and to the members of the laboratories in Valencia and Heidelberg for discussion and support. The work in the Valencia laboratory is supported by grants from the Spanish MINECO and the European Union funds (FEDER) (BFU2010-21975-C03-01 to J.E. P.-O. and PIM2010EPA-00658 to E. Herrero), and from the Regional Valencian Government (Generalitat Valenciana—PROMETEO 2011/088).
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Jordán-Pla, A., Miguel, A., Serna, E., Pelechano, V., Pérez-Ortín, J.E. (2016). Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast. In: Devaux, F. (eds) Yeast Functional Genomics. Methods in Molecular Biology, vol 1361. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3079-1_8
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DOI: https://doi.org/10.1007/978-1-4939-3079-1_8
Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3079-1
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