Phang-Cheng Tai

Complex Interactions of Antibiotics with the Ribosome

INTRODUCTION The search for antibiotics has yielded not only many life-saving drugs, but also an even larger number of valuable reagents for the study of cell physiology. The specificity and variety of their inhibitory actions have reaffirmed Claude Bernard’s dictum: “Poisons are delicate instruments that dissect vital units”; and the ribosome has turned out to be the vital unit most frequently affected. Antibiotics have contributed to our knowledge of the ribosome both through study of their mode of action and through their use in the isolation of mutants with altered ribosomes. However we shall not review the latter literature, except to note a particularly ingenious recent use of heterozygotes carrying multiple mutations to resistance: the polarity effects in these strains showed that the genes for several ribosomal proteins (and for factor EF-G) are linked in a single operon (Nomura and Engbaek 1972). It might also be noted that in studies of eukaryotic cells the selective action of cycloheximide on cytoplasmic ribosomes, and that of chloramphenicol on mitochondrial ribosomes, has been indispensable for recognizing that the latter make only a fraction of the mitochondrial proteins (Ashwell and Work 1970). The early studies of protein synthesis with synthetic messengers revealed the microcycle of chain elongation but not the macrocycle of initiation and release. Hence the first anti-ribosomal antibiotics to be understood were those that can act on elongating ribosomes. Many others yielded equivocal results until the availability of a natural messenger, viral RNA, made it possible to detect specific interactions with initiating...

Effect of streptomycin on the response of Escherichia coli ribosomes to the dissociation factor

Abstract Ribosomes recovered from cells of Escherichia coli treated with streptomycin are impaired in their response to the dissociation factor. This effect of Str evidently depends on a direct action on the ribosome and not on stabilization of a complex with normal ligands. Thus, such Str-ribosomes lack firmly bound transfer RNA; treatment with puromycin does not remove the resistance to dissociation; and similar resistance is produced when free ribosomes (in the absence of normal ligands) are exposed to Str in buffer and then washed. The impairment of dissociation of Str-ribosomes in cells is evidently incomplete, for these ribosomes maintain a reduced polysome level by engaging in cyclic abortive reinitiation (see preceding paper, Wallace & Davis, 1973) and this process requires formylation (and hence presumably dissociation). Str-inhibited dissociation may be the limiting step in this reinitiation, for the polysome level is much lower in Str-treated cells of strain W than in those of K12, and Str impairs dissociation much more with ribosomes of the former strain.

Transfer RNA content of runoff and complexed ribosomes of Escherichia coli

Abstract Runoff ribosomes extracted from Escherichia coli and washed in a buffer with 10 m m -Mg2+ contain 1 to 2 equivalents of tRNA. However, this tRNA appears to be loosely and non-specifically bound, for it is readily displaced at 37 °C (but not at 0 °C) by added tRNA, and it is largely washed off by buffer containing 3 m m -Mg2+ (which preserves the integrity of the 70 s ribosomes) or by zonal centrifugation. Moreover, runoff in vitro (at 10 m m -Mg2+) yields 70 s ribosomes with no tRNA. It thus appears that runoff ribosomes are normally free of tRNA, though they can adsorb it under certain conditions; hence the integrity of the ribosome does not depend on the binding of tRNA. Complexed ribosomes, in contrast, carry two equivalents of tRNA that is not readily washed off or displaced. At 10 m m -Mg2+ these ribosomes also carry 1 to 2 additional equivalents of loosely bound tRNA. In free ribosomes the loosely bound tRNA may be located in the normal A and P binding sites, but in complexed ribosomes it must be located elsewhere since these sites are occupied.

Impaired initiation complex formation on ribosomes treated with colicin E3

Abstract Ribosomes treated with colicin E3 are impaired, when tested with phage R17RNA as messenger, in amino acid incorporation, binding of fMet-tRNA, and formation of fMet-puromycin. Studies with subunits showed that formation of the 30S initiation complex is inhibited. The results are consistent with the recent findings that the “E3-fragment” (3′ end of 16S RNA) binds to 30S protein S1, and that both are involved in initiation on natural messenger.

Approaches to the Biochemistry of Protein Secretion in Bacteria

Studies in this laboratory on the mechanism of protein secretion in bacteria are reviewed. In Bacillus subtilis the membrane complexed with ribosomes and the free membrane were found to differ extensively in protein composition. A 64 Kd protein in the former is almost certainly involved in secretion, since it is protected by the ribosomes from interacting with protease or with antibody. This protein is also present in the cytoplasm as a complex with three additional proteins, which sediments with the 70S ribosome fraction; its relation to the ribosomes, and to the initiation of protein secretion, is under investigation. In addition, the membrane fraction associated with polysomes contains a 38 Kd protein that appears to be involved in secretion, since it remains with the polysomes after solubilization of the membrane with Triton.