Effect of spectinomycin on polypeptide synthesis in extracts of Escherichia coli

Abstract

Spcotinomycin, a dibasic aminoglycoside antibiotic, inhibits protein, synthesis reversibly on Escherichia coli ribosomes. Its effect depends strikingly on the nucleotide composition of the messenger: inhibition is completely absent with poly U or poly A; it becomes increasingly pronounced with increasing C or G content in mixed polymers; and it is almost complete with poly I. The degree of inhibition becomes maximal within a relatively narrow range of increasing Spc§ concentration. Moreover, one-step mutants with a highly resistant 30 s ribosome subunit are readily isolated. These properties suggest that the drug acts on only a single site on this subunit. In contrast to streptomycin, however, which also acts on the 30 s subunit, Spc does not cause detectable misreading in translation, and its action in vitro is independent of Mg 2+ concentration over a wide range. Spc does not appear to influence directly the initiation, recognition, peptideforming, or termination steps in polypeptide synthesis. Moreover, when ribosomes are preloaded with peptidyl-tRNA Spc does not affect amino acid transfer for the first few minutes after its addition; and evidence is presented that this lag depends on having the ribosomes reach the susceptible state, rather than on a slow interaction of Spc with some component of the system. These findings suggest that Spc blocks some aspect of the complex translocation process in the cycle of amino acid transfer.