Synthesis of a Chemically Reactive Analog of the Nonsense Codon U-G-A. Its Reaction with Ribosomes of Escherichia coli

Abstract

Nitrophenylated 5'-uridylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated Pu-G-A. After reduction of the nitrophenyl moiety and subsequent bromoacetylation, a 5'-bromoacetamido-phenyl-phosphorylated U-G-A was obtained, which could be used as an affinity label for the ribosomal binding site of the nonsense codon. If freshly prepared active ribosomes were employed in the incubation mixtures, the U-G-A analog reacted exclusively with one protein, which is tentatively identified as protein S18. Exposure of ribosomes to low temperatures gave rise to a reaction of the U-G-A label with another protein, which was identified as protein S4, the ram gene product. The results of the affinity labeling experiments with the chemically reactive U-G-A derivative are very similar to that obtained with a corresponding derivative of the initiation codon A-U-G (O. Pongs and E. Lanka (1975) Proc. Natl. Acad. Sci. U.S.A. 75, 1505-1509), which suggests that 70S ribosomes have one preferential codon binding site.