Inhibition of peptidyl-sRNA binding to ribosomes by pactamycin

Abstract

We have described ongoing studies designed to elucidate the mechanisms of action of the cytotoxic antibiotics PM and NCS.PM selectively blocks polypeptide chain initiation in eukaryotes by two effects, which may be related: (1) the joining of the 60S ribosomal subunit to the smaller initiation complex is interfered with. This action can be overcome by adding excess of a factor, obtained by DEAE-cellulose chromatography of a crude initiation factor preparation, that is normally required for joining activity; (2) under conditions where joining is not prevented, there is still a block in the formation of peptides beyond the initial dipeptide. These data can be explained by a model of PM action in which PM does not interfere with the ribosomal joining reaction per se, but prevents the release and reuse of the joining factor, and in so doing, blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome. It is possible that the block in tripeptide formation is at the level of the use of the ribosomal A-site. The selectivity of PM for polypeptide initiation may be determined by the availability of its binding site only at any early stage in initiation.NCS has been found to inhibit thymidine incorporation into DNA in vivo and to place single stranded nicks in DNA both in vivo and in vitro. While it is premature to relate the cutting of DNA by NCS in vitro to these and other effects of the drug in vivo, the test tube reaction is interesting in its own right and has revealed information on the molecular requirements for the cutting reaction. Mercaptoethanol or some other sulfhydryl compound and a pH of 7.5 are required for maximal cutting. DNA strand scission by NCS produces 5′-phosphoryl end groups on all four deoxyribonucleotides, although in preliminary experiments dGMP and TMP appear to be favored. From protection experiments, it appears that thymidylic acid in DNA is required for interaction between DNA and NCS. How these effects of NCS are related, if at all, to the block at G2 of the cell cycle (45) remains to be elucidated.