Hydroxyl Radical Footprinting in vivo: Mapping Macromolecular Structures with Synchrotron Radiation
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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 914342
- Report Number(s):
- BNL-78910-2007-JA; NARHAD; TRN: US0802853
- Journal Information:
- Nucleic Acids Res., Vol. 34, Issue 8; ISSN 0305-1048
- Country of Publication:
- United States
- Language:
- English
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