Ribosomal Assembly Influenced by Growth in the Presence of Streptomycin

Abstract

Translational leakiness (i.e., nonspecific suppression) of nonsense mutants of bacteriophage T4 is increased in cells of certain streptomycin-resistant strains previously grown in the presence of streptomycin. Con Under controlled conditions, streptomycin binds directly and specifically to naked 16S RNA, whatever its origin, and the drug's failure to bind to 30S subunits derived from streptomycin-resistant cells is apparently due to a masking of the 16S specific site(s) by the mutated S12 protein (1). Moreover, 16S RNA bound to streptomycin fails to reconstitute in vitro biologically active 30S subunits. Obviously a similar total failure to assemble active ribosomes does not occur in vivo since streptomycin-resistant strains grow normally in the presence of streptomycin. It is possible, however, that the drug might be able to interfere in a limited way with the proper assembly, resulting in functionally altered ribosomes.With these considerations in mind, we have now reexamined an unpublished observation of some years ago, which we were unable to explain at that time. In studying phenotypic suppression of T4 nonsense mutants, we observed that streptomycin programs the suppression ability of a strA40 host cell before suppression actually occurs. It may be noted that such a mechanism is not peculiar to phage infection, but that it becomes evident only with phage because it is possible to separate operationally host programming during growth before infection from actual suppression, which occurs during phage propagation.Our investigation suggests that streptomycin does indeed interfere with normal ribosomal assembly when a responsive S12 mutation is present in the genome and, as a result of this interference, a functionally altered ribosomal particle is assembled. STRAINS AND MEDIAThe bacterial strains tested for their ability for nonspecific, ribosomal suppression (measured as translational leakiness of nonsense mutations) are derived from the same Escherichia coli B strain Li (argF40, argRl1, no detectable tRNA-suppressors specific for nonsense codons). strA mutant alleles were introduced by P1 transduction: strA40 is a streptomycirresistant, moderately restrictive allele permitting expression of phenotypic suppression by streptomycin of argF40 amber (our collection number: L1-431). strAl is the most restrictive streptomycin-resistant allele we have isolated in E. coli B. It gives no detectable suppression by streptomycin of the argF40 amber (our collection number: L1-401). Strain L190 is a derivative of Li carrying a strA allele of the drugD type (3) which became streptomycin-independent through a second site mutation. Its phenotype is streptomycin or paromomycin resistant when the two drugs are used separately, but it is sensitive to a mixture of them. The X lysogens are obtained by infecting the Li strains with XC1857. They are induced at 420 but not at 300. The indicator strain used for titrating T4 wild-type phage and UGA mutants is CAJ68 (suuGA+).Cell density is calculated from the optical density (...

The Effects of Streptomycin or Dihydrostreptomycin Binding to 16S RNA or to 30S Ribosomal Subunits

“…However, it is seen in the table that misreading is high also with the 30S subunit exposed to H2Sm (about 70% of that with Sm-30S subunit). This result suggests that contact with the drug produces an alteration in the 30S subunits which persists even after the drug has been removed, in agreement with published in vivo observations (12)(13)(14) (1974) and H2Sm in a test independent from the use of labeled drugs. We had already shown (1) that Sm prevented 16S RNA and 30S proteins from E. coli B from reconstituting active 30S particles, and that this inhibition persisted even after exhaustive dialysis of the 16S RNA-Sm complex.…”

“…Dialysis easily removes dHSm from either 16S RNA or 30S subunits, with the effect on 16S RNA being readily reversed (reconstitution inhibition is completely relieved after exhaustive dialysis), while those effects on the 30S subunit persist after removal of the drug ([3H]H2Sm is not detectable, yet the ribosomes still misread as if they were in the presence of the drug). A similar persistence of drug effects has also been observed in vivo in two instances: (a) pregrowth in the presence of Sm, H2Sm, or paromomycin confers on a strA4O strain the ability to suppress phage nonsense mutations by ambiguity, although the drug is no longer present at the moment of infection (12); and (b) as "phenotypic masking" in drug dependent strains that require either Sm or paromomycin for growth but are sensitive to these drugs used together or to either one after previous growth in the other (13,14). In both cases, extracted ribosomes are shown: (1) to misread, although free of the drug (which was added during pregrowth); and (2) to be restored to normal accuracy by high salt treatment.…”

Aminoglycoside uptake and mode of action—with special reference to streptomycin and gentamicin

“…The postulated mode of action of spectinomycin bears a large number of similarities to streptomycin (Davis et al, 1974). (ii) The binding of streptomycin to ribosomes is reversible (Chang & Flaks, 1972) and does not irreversibly inactivate the ribosomes (although it may influence subsequent in-vitro misreading assays- Garvin, Rosset & Gorini, 1973). This rules out a mechanism of lasting ribosomal damage such as seen for virginiamycin M, an antibiotic that binds reversibly to ribosomes and leaves them inactive even after removal (Parfait & Cocito, 1980).…”

Collective evolution and the genetic code

“…Given an alien gene, the host-alien gene system undergoes a cyclical dynamical process leading to full utilization of the new gene. In one part of the cycle, the host detunes its own code for purposes of recognizing the alien code; an example of such a detuning process has been documented in streptomycindependent mutants (20,21) and ribosomal ambiguity mutants (22,23) in bacteria. In the other part of the cycle, the alien gene codons are mutated to conform to the host code.…”