Synthesis of a new reagent, ethyl 4-azidobenzoylaminoacetimidate, and its use for RNA-protein cross-linking within Escherichia coli ribosomal 30-S subunits.
A new reagent, ethyl 4-azidobenzoylaminoacetimidate, was prepared in a four-step synthesis starting from 4-aminobenzoic acid. This compound was used to cross-link RNA with proteins within the Escherichia 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 labelled 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-labelled RNA.
D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D007096:Imidoesters / Q000138:chemical synthesis*; D046911:Macromolecular Substances; D009843:Oligoribonucleotides / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis*; D012269:Ribosomal Proteins / Q000032:analysis*; D012270:Ribosomes / Q000032:analysis*; D013055:Spectrophotometry, Infrared
D003432:Cross-Linking Reagents; D007096:Imidoesters; D046911:Macromolecular Substances; D009843:Oligoribonucleotides; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C027921:ethyl 4-azidobenzoylaminoacetimidate