Synthesis of a New Reagent, Ethyl 4‐azidobenzoylaminoacetimidate, and Its Use for RNA‐Protein Cross‐linking within Escherichia coli Ribosomal 30‐S Subunits

Abstract

A new reagent, ethyl 4-azidobenzoylaminoacetimidate, was prepared in a 4-step synthesis starting from 4-aminobenzoic acid. This compound was used to cross-link RNA with proteins within the E. coli 30-S ribosomal subunits. Following the reaction of the imidoester function with protein NH2 groups, photoactivation of the azide binds the other end of the reagent to RNA. The cross-linked proteins were labeled with 125I and identified by bidimensional gel electrophoresis. Proteins S3, S4, S5, S7, S9, S17, S18 and, in a lower and more variable yield, S12, S13, S14 and S16 were bound to 16-S RNA. These results were confirmed by isolating cross-linked protein-oligonucleotide complexes from 30-S subunits containing 32P-labeled RNA.