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

Abstract

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.

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

“…Numerous reagents, including protein nucleases (Walczak et al 1996;Grover et al 2011;Siegfried et al 2011), alkylating chemicals such as dimethyl sulfate (DMS) (Wells et al 2000;Tijerina et al 2007;Cordero et al 2012a), and hydroxyl radicals (Adilakshmi et al 2006;Das et al 2008;Ding et al 2012), have been leveraged to modify or cleave RNA in a structure-dependent manner. Protection of nucleotides from modification, typically signaling the formation of base pairs, can guide manual or automatic secondary structure inference (Mathews et al 2004;Mitra et al 2008;Vasa et al 2008).…”

RNA folding in living cells

“…To date, several chemical reagents, which are sensitive to secondary and/or tertiary structure, have been used for probing RNA structure in vivo (reviewed in ref. [118][119][120][121][122]. DMS is the most successfully used chemical to probe RNA structure in a variety of organisms, ranging from bacteria to eukaryotes.…”

“…Lastly, hydroxyl radical footprinting can be applied to probe RNA tertiary structure and intermolecular interfaces. 118 In brief, X-rays from a high flux synchrotron generates hydroxyl radicals directly inside cells, which in turn can abstract a hydrogen atom from the ribose and initiate cleavage of the RNA backbone. 118 By this, hydroxyl radical cleavage correlates with the solvent accessibility of the backbone, providing information at nucleotide resolution.…”

“…118 In brief, X-rays from a high flux synchrotron generates hydroxyl radicals directly inside cells, which in turn can abstract a hydrogen atom from the ribose and initiate cleavage of the RNA backbone. 118 By this, hydroxyl radical cleavage correlates with the solvent accessibility of the backbone, providing information at nucleotide resolution. Notably, the modification or cleavage sites are then mapped by primer extension of total RNA extracts, revealing information about RNA structure and RNA-protein interaction in vivo.…”

The Cech Symposium: A celebration of 25 years of ribozymes, 10 years of TERT, and 60 years of Tom

“…In short, assembly occurs by alternating series of RNA conformational changes and protein binding events that create a folding landscape devoid of kinetic traps. Also studying the assembly of the ribosome, Sarah Woodson (Johns Hopkins University) used time-resolved footprinting to reveal that the assembly of the 30S particle relies on local RNA-RNA interactions and on the binding of ribosomal proteins in a stochastic manner (Adilakshmi et al 2006). Complementing the talks on ribosomal assembly, Scott Strobel (Yale University) presented a detailed structural and enzymatic investigation of the ribosomal petidyl-transferase mechanism.…”