Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Modulation of Decoding Fidelity by Ribosomal Proteins S4 and S5

Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA.     

Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA.

We have used chemical modification to examine the conformation of 23 S rRNA in Escherichia coli ribosomes bearing erythromycin resistance mutations in ribosomal proteins L22 and L4. Changes in reactivity to chemical probes were observed at several nucleotide positions scattered throughout 23 S rRNA. The L4 mutation affects the reactivity of G799 and U1255 in domain II and that of A2572 in domain V. The L22 mutation influences modification in domain II at positions m5U747, G748, and A1268, as well as at A1614 in domain III and G2351 in domain V. The reactivity of A789 is weakly enhanced by both the L22 and L4 mutations. None of these nucleotide positions has previously been associated with macrolide antibiotic resistance. Interestingly, neither of the ribosomal protein mutations produces any detectable effects at or within the vicinity of A2058 in domain V, the site most frequently shown to confer macrolide resistance when altered by methylation or mutation. Thus, while L22 and L4 bind primarily to domain I of 23 S rRNA, erythromycin resistance mutations in these ribosomal proteins perturb the conformation of residues in domains II, III and V and affect the action of antibiotics known to interact with nucleotide residues in the peptidyl transferase center of domain V. These results support the hypothesis that ribosomal proteins interact with rRNA at multiple sites to establish its functionally active three-dimensional structure, and suggest that these antibiotic resistance mutations act by perturbing the conformation of rRNA.     

Mutational alterations in 50 proteins of the Escherichia coli ribosome.

Summary A strain of Escherichia coli, VT, which spontaneously gives rise to mutations in many ribosomal proteins, has been used in conjunction with chemical mutagenesis and varying the subsequent incubation temperature to select mutants which have alterations in every ribosomal protein amenable to analysis of 70 S proteins on two-dimensional polyacrylamide gels under standard conditions. Alterations have been detected in 50 ribosomal proteins, namely in 20 from the small and in 30 from the large subunit. This is the most complete set of mutants with altered ribosomal proteins described so far. The difficulty until recently in obtaining mutations in most ribosomal proteins arises not because they are lethal, as has often been supposed, but because of the lack of a suitable selection heretofore.Communicated by E. Bautz     

Analysis of ribosomal proteins in streptomycin resistant and dependent mutants isolated from streptomycin independent Escherichia coli strains

Summary Mutants resistant to (Str-R) or dependent on streptomycin (Str-D) were isolated from several streptomycin independent (Str-I) strains of Escherichia coli. From 90 of these mutants ribosomes were isolated and the ribosomal proteins analyzed by two-dimensional polyacrylamide gel electrophoresis. The results which are summarized in Tables 1-4 led to the following conclusions:a) The phenotype (Str-R or Str-D) of the mutants isolated from the Str-I strains strongly depends on the parental strain. b) No other ribosomal proteins than S4, S5 and S12 seem to be altered by mutations leading to dependence on, independence from or resistance to streptomycin. c) The S4 proteins of the analyzed mutants belong to three groups. The ratio between the groups depends more on the origin of the mutants than on their phenotype. d) Eight new types of altered S4 proteins were detected. It is very likely that many, if not all, of the altered S4 proteins originated by frame shift mutations. e) Some of the mutants differ from the wild type by alterations in three ribosomal proteins (S4, S5 and S12). The alteration in one protein, S4, apparently compensates for that in another protein, S5, in such a way that the original phenotype is expressed. These mutants are therefore an excellent tool for studies at the molecular level on the interaction of ribosomal components within the particle.     

A strain of Escherichia coli which gives rise to mutations in a large number of ribosomal proteins

Summary A strain of E. coli K12 has been isolated which gives rise to mutations in a large number of ribosomal proteins. Mutant VT, which was derived from A19, shows a novel type of streptomycin dependence and has an altered ribosomal protein S8. Streptomycin-independent isolates from mutant VT contain a great variety of changed proteins on two-dimensional polyacrylamide gels. 120 revertants screened in this way have changes in thirteen 30S proteins and fifteen 50S proteins. Several mutants were found in which additional proteins are present on the ribosome. Further, there is one instance of a ribosomal protein (L1) being absent, and one of apparent doubling of a ribosomal protein (L7/12). The unique properties of mutant VT probably are the result of the altered S8.     

A functional interaction between ribosomal proteins S7 and S11 within the bacterial ribosome

In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.     

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.     

Alternative conformers of 5S ribosomal RNA and their biological relevance

Different conformational states of Escherichia coli 5S ribosomal RNA that may participate in protein biosynthesis have been either detected experimentally or predicted on the basis of phylogenetic sequence comparisons. The A conformer exists in a high-salt form (AH) that binds ribosomal proteins and assembles into the 50S subunit and in a low-salt form (AL), of uncertain biological relevance, that binds at least one ribosomal protein and differs in tertiary structure from the AH form. Experimentally, the AH form has been investigated comprehensively and the AL form partially. There is also a B conformer that exhibits an altered secondary structure and does not assemble with ribosomal proteins. For this conformer exhibits an altered secondary structure and does not assemble with ribosomal proteins. For this conformer to be functionally active, it must be both discrete and universal among 5S RNAs. Here, we examine its structure by employing single and double strand specific ribonucleases and nucleotide-specific chemical reagents. We demonstrate that the B form exhibits a secondary structure only a part of which is both universal and conformationally homogeneous, and we conclude, therefore, that the whole B form cannot participate in protein biosynthesis. We note, however, that progressive structural changes occur during the transitions AH----AL----B and provide evidence that the structural alteration during the transition AH----AL may be universal, which reinforces the view that the AL form is of biological relevance.     

Identification of the amino acid residues of proteins S5 and S8 adjacent to each other in the 30 S ribosomal subunit of Escherichia coli.

When Escherichia coli 30 S ribosomal subunits are reacted with protein-protein bifunctional reagents, a number of protein pairs as well as aggregates containing three or more ribosomal proteins are formed. In the present study we have purified one of the protein pairs obtained by reaction of 30 S ribosomal subunits with either radioactive or nonradioactive dimethylsuberimidate. Following molecular weight determination and ammonolysis, the pair was shown to consist of ribosomal proteins S5 and S8. The "native" structure of the complex was surmised from its capacity to be reconstituted into a biologically active 30 S ribosomal subunit. From peptide maps and primary structure determination of various peptides it was demonstrated that the cross-linking bond between ribosomal proteins S5 and S8 involves primarily the residues Lys-93 of protein S8 and the COOH-terminal lysine (Lys-166) of ribosomal protein S5. This result is substantiated by the finding that a mutant carrying an altered S5 lacking the COOH-terminal lysine yields a greatly reduced amount of S5-S8 cross-link. In addition to the points of cross-linking it was found that Lys-30, Lys-68, and Lys-86 of S8 and Lys-5 of S5 react with dimethylsuberimidate, indicating that these residues are available for reaction and suggesting their topographical localization on the ribosomal surface.     

Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants.

Summary 26 cold-resistant revertants of a cold-sensitiveEscherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis. It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered S8 than the original mutant and two revertants regained the wild-type form of S8 and, in addition, possess alterations in protein L30. The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant.The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site. The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is:aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....THe L30 mutation impairs growth and ribosomal assembly at 20°C and is therefore the first example of a mutant with a defined 50S alteration that has (partial) cold-sensitive ribosome assembly. A double mutant was constructed which possesses both the S8 and the L30 mutations. It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation. Thus the L30 mutants might have possibly arisen from the original S8 mutants first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.     

Compensatory evolution reveals functional interactions between ribosomal proteins S12, L14 and L19

Certain mutations in S12, a ribosomal protein involved in translation elongation rate and translation accuracy, confer resistance to the aminoglycoside streptomycin. Previously we showed in Salmonella typhimurium that the fitness cost, i.e. reduced growth rate, due to the amino acid substitution K42N in S12 could be compensated by at least 35 different mutations located in the ribosomal proteins S4, S5 and L19. Here, we have characterized in vivo the fitness, translation speed and translation accuracy of four different L19 mutants. When separated from the resistance mutation located in S12, the three different compensatory amino acid substitutions in L19 at position 40 (Q40H, Q40L and Q40R) caused a decrease in fitness while the G104A change had no effect on bacterial growth. The rate of protein synthesis was unaffected or increased by the mutations at position 40 and the level of read-through of a UGA nonsense codon was increased in vivo, indicating a loss of translational accuracy. The mutations in L19 increased sensitivity to aminoglycosides active at the A-site, further indicating a perturbation of the decoding step. These phenotypes are similar to those of the classical S4 and S5 ram (ribosomal ambiguity) mutants. By evolving low-fitness L19 mutants by serial passage, we showed that the fitness cost conferred by the L19 mutations could be compensated by additional mutations in the ribosomal protein L19 itself, in S12 and in L14, a protein located close to L19. Our results reveal a novel functional role for the 50 S ribosomal protein L19 during protein synthesis, supporting published structural data suggesting that the interaction of L14 and L19 with 16 S rRNA could influence function of the 30 S subunit. Moreover, our study demonstrates how compensatory fitness-evolution can be used to discover new molecular functions of ribosomal proteins.     

Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA

We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS-unreactive. In this model, the balance between these two states would be influenced by proteins S4 and S12. Mutations in S12 generally cause a shift toward the unreactive conformer, and in the case of SmD and SmP ribosomes, this shift can be suppressed phenotypically by streptomycin, ram mutations in protein S4 cause a shift toward the reactive conformer, but only when tRNA is bound. This suggests that the opposing effects of these two classes of mutations influence the proof-reading process by somewhat different mechanisms.     

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

Linear ubiquitin fusion to Rps31 and its subsequent cleavage are required for the efficient production and functional integrity of 40S ribosomal subunits

The post-translational modifier ubiquitin is generated exclusively by proteolytic cleavage of precursor proteins. In Saccharomyces cerevisiae , cleavage of the linear precursor proteins releases ubiquitin and the C-terminally fused ribosomal proteins Rpl40 (Ubi1/2 precursor) and Rps31 (Ubi3 precursor), which are part of mature 60S and 40S ribosomal subunits respectively. In this study, we analysed the effects of ubi3 mutations that interfere with cleavage of the ubiquitin–Rps31 fusion protein. Strikingly, the lethal ubi3 + P77 mutation, which abolished cleavage almost completely, led to a rapid G1 cell cycle arrest upon genetic depletion of wild-type UBI3 . Under these conditions, the otherwise unstable Ubi3+P77 protein was efficiently assembled into translation-competent 40S ribosomal subunits. In contrast to the cleavage-affecting mutations, deletion of the ubiquitin moiety from UBI3 led to a decrease in 40S ribosomal subunits and to the incorporation of the 20S pre-rRNA into polyribosomes. Altogether, our findings provide additional evidence that the initial presence of the ubiquitin moiety of Ubi3 contributes to the efficient production of 40S ribosomal subunits and they suggest that ubiquitin release is a prerequisite for their functional integrity.     

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.     

Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein.

The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence of a ribosomal protein gene that affects translational ambiguity in a eucaryote. We show that the yeast omnipotent suppressor SUP44 encodes the yeast ribosomal protein S4. The gene exists as a single copy without an intron. The SUP44 protein is 26% identical (54% similar) to the well-characterized E. coli S5 ram protein. SUP44 is also 59% identical (78% similar) to mouse protein LLrep3, whose function was previously unknown (D.L. Heller, K.M. Gianda, and L. Leinwand, Mol. Cell. Biol. 8:2797-2803, 1988). The SUP44 suppressor mutation occurs near a region of the protein that corresponds to the known positions of alterations in E. coli S5 ram mutations. This is the first ribosomal protein whose function and sequence have been shown to be conserved between procaryotes and eucaryotes.     

Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5 in Bacillus subtilis

To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in a ram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.