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Nucleic Acids Research 2006 34(8):e64; doi:10.1093/nar/gkl291
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Published online 8 May 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Methods Online

Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation

Tadepalli Adilakshmi, Richard A. Lease and Sarah A. Woodson*

T.C. Jenkins Department of Biophysics, Johns Hopkins University 3400 North Charles Street, Baltimore, MD 21218, USA

*To whom correspondence should be addressed. Tel: +1 410 516 2015; Fax: +1 410 516 4118; Email: swoodson{at}jhu.edu

Received March 8, 2006. Revised April 6, 2006. Accepted April 6, 2006.

We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA–protein complexes with nucleotide resolution in living cells.

Present address: Richard A. Lease, Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA


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