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The functional peculiarities of ram mutants correlate with an observed alteration in chromatographic mobility of P4a, a specific protein of the 30S ribosomal subunit. This finding is supported by ribosomnal reconstitution experiments. These facts, together with the known location of the ram mutational site in the vicinity of other 30S genetic determinants, suggest that ram is the structural gene for P4a.The known contrasting roles of ram and strA in determining translational efficiency require that the function of P4a should be explained in relation to PlO (the 30S-subunit protein defined by strA). One consequence of altering P4a, a key protein in ribosome assembly, might be to change the interaction of PlO with the 30S subunit. The functional interrelationship of P4a and PlO is discussed in terms of the possible roles of these two proteins in regulating access of tRNA molecules to the decoding site.A mutation isolated by selection for increased translational ambiguity in Escherichia coli B was found to alter the 30S ribosomal subunit. Strains bearing this lesion, which maps in the strA region, have ribosomes that allow translation of synthetic mRNAs with a level of misreading significantly higher than ribosomes from parental strains; this capacity was shown to be a property of the mutant 30S subunit by exchange of 30S and 50S particles from mutant and parental ribosomes. The new mutation was accordingly called ram, for ribosomal ambiguity (1).A more general interpretation of the nature of ram mutants, however, emerges from a consideration of the procedure used in their selection. The parent was an argF amber mutant whose nutritional block was incomplete due to limited or "leaky" translation through the nonsense codon (2). In the first step of the selection, the strain was made strictly auxotrophic by introducing strAl, a mutant allele of the gene specifying the 30S ribosomal protein P10 (3), known to restrict intrinsic nonsense leakiness (4). In the second step, spontaneous mutants were sought in which the nutritional leakiness of the original parent reappeared. Given the nature of the selection, it is quite possible that ram determines a 30S ribosomal component that does not, in itself, govern translational ambiguity, but which in some way opposes the translational restriction imposed by the strA mutation.This view is supported by the fact that the ram/strA interaction is not limited to translational ambiguity. A further aspect of the phenotype, which seems more general, is that the ram mutation reverses the restriction imposed by strA alleles on the suppression efficiency of mutated tRNAs (4, T). This reversal cannot be attributed to increased ambiguity, as demonstrated in the case of missense suppression; if ram were to introduce ambiguity, a further reduction, rather than an increase, in suppression efficiency would be expected, since the insertion of a specific amino acid is required to correct a missense mutation and restore functional activity to the gene product.The contrasting influence on tra...

Evidence is presented suggesting that streptomycin binds to 16S RNA or to 30S ribosomal subunits at the same topographical site located on the RNA chain. The equally bactericidal dihydrostreptomycin binds to the same site as streptomycin but with lower affinity. The effect of drug binding to 16S RNA (measured by reconstitution inhibition) is readily reversible, while that of drug binding to 30S subunits (measured by misreading) persists after removal of the drug. Binding of the drug is not a necessary and sufficient reason for killing.The earlier publication (1) that the site of streptomycin (Sm) binding to the 30S ribosomal subunit could be the 16S RNA may be criticized from several viewpoints. On the one hand, according to stoichiometry established for dihydrostreptomycin (H2Sm)-30S binding (2, 3), too much Sm was bound; on the other hand, in view of the potential of nucleic acid for binding the drug, too little Sm was bound. Furthermore, it is unexpected that Sm should have any specificity at all (for example, stable binding to 16S and no binding to 23S RNA).Experiments to answer these criticisms were designed with every attempt being made to relate binding and physiology. We report here the results of these experiments, which (1) confirm the role of 16S RNA as drug binding site; (2) demonstrate that Sm and H2Sm are quantitatively different with regard to binding (nevertheless they remain indistinguishable with respect to bactericidal action); (3) confirm that misreading is a hysteretic effect of either Sm or H2Sm and does not require the physical presence of the drug; and (4) Preparation of Ribosomal Components. 70S ribosomes were prepared by sedimentation (270,000 X g) of the DNasetreated lysate obtained by grinding frozen cells with alumina and removing cell debris. Subunits were dissociated by dialysis in low Mg buffer (B2+) followed by large scale zonal centrifugation in a Beckman TI-15 rotor. 16S RNA was isolated, by phenol extraction, directly from the 30S subunits obtained through the zonal centrifugation. The 30S subunits were absorbed on a DEAE-cellulose column; washed with 0.25 M NH4Cl (2 column volumes), and eluted with 0.5 M NH4Cl. These subunits, which fail to produce ethanol-soluble radioactivity when tested for RNase activity on [3H]poly(uridylic acid), were the source of the total 30S protein fraction, prepared by mixing with an equal volume of [4][5][6][7][8] solution. 30S subunits from the isolated 16S RNA (3H-labeled 16S RNA extracted from a [3H]uridine grown culture, was used whenever possible) and total protein components were reconstituted by incubation in RC+ buffer following the usual procedure. For details of all manipulations concerning the ribosomal components see refs. 1 and 8.The physical characteristics of each 30S subunit or 16S RNA component preparation, and of pellets obtained from each reconstitution experiment, were determined by absorbance and/or radioactivity measurements made upon each aliquot of a fractionated sucrose density gradient (5-20% sucrose in B2+) pre...

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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 (...