Synthesis of a chemically reactive analog of the initiation codon: its reaction with ribosomes of Escherichia coli.

Nitrophenylated 5'-adenylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated pA-U-G. After reduction and subsequent bromoacetylation, an A-U-G analog was obtained, which could be used as an affinity label for the ribosomal A-U-G-binding site(s). After incubating the A-U-G affinity label with 70S ribosomes, 30S subunits programmed for initiation-factor-dependent fMet-tRNAMetf binding were obtained. Hence, the A-U-G analog had irreversibly reacted at the ribosomal decoding site. Initiation complexes which were formed with the labeled 30S subunits were puromycin-resistant. Furthermore, GTP hydrolysis, necessary for proper accommodation of initiator tRNA at the ribosomal donorsite, did not function in these complexes. These data indicate that immobilization of A-U-G at the decoding site of the ribosome allows factor-dependent initiator tRNA binding, but impairs accommodation at the donor site. The ribosomal protein(s) to which A-U-G was covalently bound at the decoding site were identified by polyacrylamide gel electrophoresis in the presence of urea or sarkosyl. The predominant affinity-labeled protein was found to be protein S18. Variation of the incubation conditions of the affinity-labeling reaction leads to attachment of A-U-G label to another ribosomal protein, S4, the ram gene product.