A novel approach to introduce site-directed specific cross-links within RNA-protein complexes. Application to the Escherichia coli threonyl-tRNA synthetase/translational operator complex.

Abstract

We describe a methodology which allows the introduction of a photoactivatable azido group at specific internal positions of any RNA in order to identify the neighboring elements of an interacting protein. The first step involves site-directed modification of the target RNA with an antisense oligodeoxyribonucleotide bearing, at its 3' or 5' phosphate, a 4-[-N-(2-chloroethyl)-N-methylamino]benzylmethylamino group. Position N7 of a guanine residue located in the close vicinity of the hybrid is the main target for alkylation. The antisense oligodeoxyribonucleotide is then removed by acidic pH treatment and a photoreactive reagent (2,4-dinitro-5-fluorophenylazide) is condensed to the modified nucleotide. This method was used to induce specific cross-links between Escherichia coli threonyl-tRNA synthetase and the leader region of threonyl-tRNA synthetase mRNA, which is involved in translational feedback regulation. Control experiments revealed that the modification affects neither the structure of the mRNA nor the interaction with the enzyme. More than 50% of the modified mRNA complexed with threonyl-tRNA synthetase can be cross-linked to the enzyme, depending on the nucleotide modified.