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Pyrite footprinting of RNA.

Abstract：

:In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ((·)OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS(2)) can produce sufficient (·)OH to footprint DNA. The 49-nt Diels-Alder RNA enzyme catalyzes the C-C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme's active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels-Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to (·)OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop's flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated (·)OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Schlatterer JC,Wieder MS,Jones CD,Pollack L,Brenowitz M

doi

10.1016/j.bbrc.2012.07.100

subject

Has Abstract

pub_date

2012-08-24 00:00:00

pages

374-8

issue

2

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(12)01407-6

journal_volume

425

pub_type

杂志文章

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