In Vivo RNA Chemical Footprinting Analysis in Archaea
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RNA structural conformation and dynamics govern the functional properties of all RNA/RNP. Accordingly, defining changes of RNA structure and dynamics in various conditions may provide detailed insight into how RNA structural properties regulate the function of RNA/RNP. Traditional chemical footprinting analysis using chemical modifiers allows to sample the dynamics and conformation landscape of diverse RNA/RNP. However, many chemical modifiers are limited in their capacity to provide unbiased information reflecting the in vivo RNA/RNP structural landscape. In the recent years, the development of selective-2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology that uses powerful new chemical modifiers has significantly improved in vitro and in vivo structural probing of secondary and tertiary interactions of diverse RNA species at the single nucleotide level.
Although the original discovery of Archaea as an independent domain of life is intimately linked to the technological development of RNA analysis, our understanding of in vivo RNA structural conformation and dynamics in this domain of life remains scarce.
This protocol describes the in vivo use of SHAPE chemistry in two evolutionary divergent model Archaea, Sulfolobus acidocaldarius and Haloferax volcanii.
Key wordsArchaea RNA RNP Chemical footprinting SHAPE NMIA 1m6 1m7 Sulfolobus acidocaldarius Haloferax volcanii
We are grateful to Dr. Tilman Heise (University of Regensburg) for comments and suggestions. We would like to thank our colleagues from the Chair of Biochemistry III and Biochemistry I for sharing materials, equipment, and discussion. Thanks to Prof. Dr. Sonja-Verena Albers (University of Freiburg) and Prof. Dr. Thorsten Allers (University of Nottingham) for sharing strains and protocols. Work in the Ferreira-Cerca laboratory is supported by the Chair of Biochemistry III “House of the Ribosome”-University of Regensburg, by the DFG-funded collaborative research center CRC/SFB960 “RNP Biogenesis: Assembly of Ribosomes and Non-ribosomal RNPs and Control of Their Function” (project AP1/B13), and by an individual DFG grant to S.F.-C. (FE1622/2-1 Project Nr. 409198929).
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