Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

Ryan M. Hulscher, Jen Bohon, Mollie C. Rappé, Sayan Gupta, Rhijuta D'Mello, Michael Sullivan, Corie Y. Ralston, Mark R. Chance, Sarah A. Woodson

Research output: Contribution to journalArticle

Abstract

The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.

Original languageEnglish (US)
JournalMethods
DOIs
StateAccepted/In press - Jan 20 2016

Keywords

  • 4-Thiouridine
  • Dimethylsulfate
  • Hydroxyl radical footprinting
  • Ribosome assembly
  • RNA structure
  • Synchrotron X-ray beamline

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Hulscher, R. M., Bohon, J., Rappé, M. C., Gupta, S., D'Mello, R., Sullivan, M., Ralston, C. Y., Chance, M. R., & Woodson, S. A. (Accepted/In press). Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting. Methods. https://doi.org/10.1016/j.ymeth.2016.03.012