Donald T. Dubin

The sequence of some effects of streptomycin in Escherichia coli

The effects of streptomycin on a number of biochemical parameters in growing cultures of Escherichia coli have been studied. Using low concentrations of drug and radioactive tracer techniques we have timed these effects relative to each other and to the killing action of the drug. The earlier effects noted were: first, an immediate (primary) uptake of streptomycin; next, an acceleration of K efflux and a transient stimulation of net RNA synthesis; and then, inhibition of protein synthesis and loss of viability. Later effects were: a further (secondary) uptake of streptomycin, increased permeability to a β-galactoside, impairment of respiration, inhibition of RNA and DNA synthesis and RNA breakdown and nucleotide excretion. The present findings cannot be integrated into a definitive theory on the action of streptomycin, but they do emphasize the importance of effects on membrane integrity and on the synthesis of RNA and protein.

Minor nucleotide composition of ribosomal precursor, and ribosomal, ribonucleic acid in Escherichia coli

Abstract 1. The minor nucleotide composition of the “ribosomal precursor” RNA of Escherichia coli has been compared with that of mature ribosomal RNA. When cells were treated with chloramphenicol, or with streptomycin, or when a “relaxed” auxotroph was starved of a required amino acid, the newly synthesized, “ribosomal precursor”, 16-S RNA (centrifugally purified) was found to be undermethylated by 70–80 %, and 23-S RNA by 40–45 %. Nucleotide analyses showed that the 23S precursor component was about equally deficient in all normal methylated nucleotides, except for an O′- ribose-methylated nucleotide, whose accumulation was more drastically affected. The 16S precursor component had virtually no methylated nucleotides, except for approximately one 5-methylcytidylic acid residue per molecule. These results are in accord with the idea that undermethylation causes previously observed centrifugal and chromatographic abnormalities of ribosomal precursor RNA. 2. Chloramphenicol treatment was also found to inhibit the formation of pseudouridylic acid in newly synthesized ribosomal RNA. 3. No differences in ribosomal RNA methylation patterns were noted among strains B, W, or K12, or between streptomycin-sensitive and streptomycin-resistant strains. 4. A model is presented whereby certain “structural” ribosomal proteins also function as ribosomal RNA-modifying enzymes during ribosome assembly.

Effect of puromycin aminonucleoside on RNA synthesis in L cells

Puromycin aminonucleoside has recently been shown to be a potent inhibitor of RNA synthesis in L cells ( Farnham, 1965 ). In the present studies, RNA from normal and aminonucleoside-treated cells has been fractionated by sucrose density-gradient centrifugation and analyzed for base composition. At a time when aminonucleoside had produced about 50% inhibition in over-all RNA synthesis, there appeared to be a relative preservation of S-RNA‡ and M-RNA synthesis. Calculations, made on the basis of base composition analyses, indicated that whereas R-RNA synthesis in the aminonucleoside-treated cells was inhibited by about 85%, M-RNA synthesis was inhibited by about 10%.

Some effects of streptomycin on RNA metabolism in Escherichia coli

Streptomycin is known to allow considerable ribonucleic acid synthesis even after it has stopped protein synthesis in sensitive cells. This “streptomycin RNA” has now been shown to resemble chloramphenicol RNA in that (1) it has a relatively normal size distribution when purified and fractionated by means of sucrose density-gradient centrifugation ; (2) a portion of it is incorporated into abnormally slowly sedimenting particles; and (3) a portion (perhaps the same portion as in (2)) is unstable both in cells and in cell extracts. Streptomycin can also cause a net stimulation of RNA synthesis, and at low drug concentrations this stimulation precedes detectable slowing of protein synthesis. The early stimulation has been shown to involve each of the major centrifugally-separable classes of normal RNA as well as messenger RNA. In addition, the ribosomal components in this case are incorporated into normal ribosomal subunits. Similar results were obtained using very low concentrations of chloramphenicol. It is proposed that the stimulation of RNA synthesis by both drugs may be the result of inhibition of protein synthesis too slight to be detected directly. While the effects of chloramphenicol and streptomycin on RNA synthesis thus appear quite similar, their effects on RNA stability differ significantly. Streptomycin-treated cells undergo a generalized RNA breakdown which contrasts with the limited breakdown to which both chloramphenicol RNA and streptomycin RNA are susceptible. The present findings are discussed with reference to the regulation of RNA synthesis as well as to the possible mechanisms of action of streptomycin.

Some abnormal properties of chloramphenicol RNA

A detailed analysis of the ribosomal components of chloramphenicol ribonucleic acid has revealed slight but definite differences from normal ribosomal ribonucleic acid. Both the 16 s and the 23 s components of chloramphenicol ribonucleic acid sedimented slightly faster through sucrose gradients, and had slightly less affinity for columns of methylated albumin-coated kieselguhr, than their normal counterparts. These differences are explainable in terms of secondary structure; chloramphenicol ribosomal ribonucleic acid may have a greater degree of ordered helical configuration than normal ribosomal ribonucleic acid.

Studies on the mechanism of action of puromycin aminonucleoside in L cells

Abstract 1. 1. Puromycin aminonucleoside preferentially inhibits ribosomal RNA synthesis in L cells while at the same time sparing DNA and protein synthesis. This effect has been examined further and, using radioactive puromycin aminonucleoside, information has been obtained regarding the metabolism of this compound. 2. 2. Equimolar concentrations of adenosine and guanosine delayed, but did not prevent, the RNA inhibitory effect of puromycin aminonucleoside. Furthermore, the utilization of exogenous [ 14 C]adenosine was inhibited to the same extent as was [ 14 C]uridine utilization. It therefore seems unlikely that puromycin aminonucleoside inhibits RNA synthesis in this system by interfering directly with purine synthesis de novo . 3. 3. The RNA synthetic capacity of puromycin aminonucleoside-treated cells began to recover only after about 20 h in drug-free medium, and the presence of adenosine did not hasten this process. This finding correlated well with the lag in the resumption of growth by puromycin aminonucleoside-treated cells. 4. 4. The DNA-dependent RNA polymerase activity from L-cell nuclei was inhibited to a small extent (20 %) by puromycin aminonucleoside. 5. 5. Experiments using radioactive puromycin aminonucleoside demonstrated that L cells metabolized significant amounts of this drug to compounds tentatively identified as phosphorylated 3′-amino-3′-deoxyadenosine derivatives. No substantial incorporation of puromycin aminonucleoside or 3′-amino-3′-deoxyadenosine into L-cell RNA could be demonstrated.

RNA-DNA hybrid formation with methyl-deficient and mature ribosomal RNA from Escherichia coli.

The effect of the presence of methylated bases in ribosomal RNA on the efficiency with which the RNA forms hybrid complexes with complementary DNA has been investigated. A comparison between methyl-deficient ribosomal RNA, from methionine-deprived and from chloramphenicol-treated Escherichia coli , and mature ribosomal RNA from untreated cells, showed no differences in hybridization efficiency. Evidence for the accumulation of messenger RNA in both methionine-deprived and chloramphenicol-treated cells was obtained.