A NOVEL-APPROACH TO INTRODUCE SITE-DIRECTED SPECIFIC CROSS-LINKS WITHIN RNA-PROTEIN COMPLEXES - APPLICATION TO THE ESCHERICHIA-COLI THREONYL-TRANSFER-RNA SYNTHETASE TRANSLATIONAL OPERATOR COMPLEX

We describe a methodology which allows the introduction of a photoacti vatable azido group at specific internal positions of any RNA in order to identify the neighboring elements of an interacting protein. The f irst step involves site-directed modification of the target RNA with a n antisense oligodeoxyribonucleotide bearing, at its 3' or 5' phosphat e, a 4-[-N-(2-chloroethyl)-N-methylamino]benz group. Position N7 of a guanine residue located in the close vicinity of the hybrid is the mai n target for alkylation. The antisense oligodeoxyribonucleotide is the n removed by acidic pH treatment and a photoreactive reagent (2,4-dini tro-5-fluorophenylazide) is condensed to the modified nucleotide. This method was used to induce specific cross-links between Escherichia co li threonyl-tRNA synthetase and the leader region of threonyl-tRNA syn thetase 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 b e cross-linked to the enzyme, depending on the nucleotide modified.