Subunit association defects in Escherichia coli ribosome mutants lacking proteins S20 and L11

The subunit association capacity of 30S and 50S ribosomal subunits from Escherichia coli mutants lacking protein S20 or L11 as well as of 50S subunits depleted of L7/L12 was tested by sucrose gradient centrifugation and by a nitrocellulose filtration method based on the protection from hydrolysis with peptidyl-tRNA hydrolase of ribosome-bound AcPhe-tRNA. It was found that the subunits lacking either S20 or L11 display an altered association capacity, while the 50S subunits lacking L7/L12 have normal association behavior. The association of S20-lacking 30S subunits is quantitatively reduced, especially at low Mg2+ concentrations (5-12 mM), and produces loosely interacting particles which dissociate during sucrose gradient centrifugation. The association of L11-lacking 50S subunits is quantitatively near-normal at all Mg2+ concentrations and produces loosely associating particles only at low Mg2+ concentrations (5-8 mM); the mechanism of their association with 30S subunits, however, or the structure of the resulting 30S-50S couples is altered in such a way as to cause the ejection of an AcPhe-tRNA molecule pre-bound to the 30S subunits in response to poly(U).     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.     

Biogenesis of mitochondria. 53

Summary As shown by gel electrophoresis analysis, E. coli mutant 219 is mutated on the gene coding for S4. This mutant and the parental strain have been studied at the permissive (30°) and the non-permissive temperature (42°) for ribosome assembly and r-protein biosynthesis.The extracts of cells grown at the non-permissive temperature were analyzed by sucrose gradients: Particles sedimenting more slowly (28S) than normal 30S accumulate while 50S precursors undergo maturation and attach to the preformed 30S subunits yielding 70S ribosomes. In addition a small but detectable amount of 30S is also synthesized at 42°. The 28S particles contain all 30S r-proteins except S1, S2 and S12; S5, S7 and S21 are present in reduced amount.The relative rate of biosynthesis of individual r-proteins was determined by pulse-labelling the cells with radioactive leucine. Individual r-proteins were purified from cell extract by the three-dimensional gel electrophoresis technique. The relative rate of biosynthesis of 50S proteins is unchanged in mutant cells grown at 42°. Only the rate of synthesis of five 30S proteins is modified by the temperature shift: S10, S13, S20 and S21 have an increased rate, while S18 is synthesized at a reduced rate. Thus in cells deficient in the assembly of 30S subunits, although the biosynthesis of a few 30S r-proteins is specifically altered, the synthesis of most r-proteins appears to be controlled in the same way as are total cell proteins.     

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.     

Multiple defects in translation associated with altered ribosomal protein L4

The ribosomal proteins L4 and L22 form part of the peptide exit tunnel in the large ribosomal subunit. In Escherichia coli, alterations in either of these proteins can confer resistance to the macrolide antibiotic, erythromycin. The structures of the 30S as well as the 50S subunits from each antibiotic resistant mutant differ from wild type in distinct ways and L4 mutant ribosomes have decreased peptide bond-forming activity. Our analyses of the decoding properties of both mutants show that ribosomes carrying the altered L4 protein support increased levels of frameshifting, missense decoding and readthrough of stop codons during the elongation phase of protein synthesis and stimulate utilization of non-AUG codons and mutant initiator tRNAs at initiation. L4 mutant ribosomes are also altered in their interactions with a range of 30S-targeted antibiotics. In contrast, the L22 mutant is relatively unaffected in both decoding activities and antibiotic interactions. These results suggest that mutations in the large subunit protein L4 not only alter the structure of the 50S subunit, but upon subunit association, also affect the structure and function of the 30S subunit.     

Regulation of gene expression at the translational level. The rescue factor reverses thermosensitive protein synthesis in N4316, a conditionally-lethal mutant of Escherichia coli defective in translation

Extracts of the conditionally-lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non-permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non-permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read-through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis. Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine-puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.     

Genetic and biochemical analysis of ribosomal proteins of minocycline-susceptible and -resistant Mycobacterium smegmatis.

A minocycline (MINO)-resistant mutant was isolated from Mycobacterium smegmatis strain Rabinowitschi. Polypeptide synthesis in the cell-free system prepared from the mutant was resistant to minocycline (MINO) because of alterated 30S ribosomal subunits. Upon two-dimensional gel electrophoresis, two proteins of 30S subunit were found to be altered. MINO resistance phenotype was transferred by mating to the recipient strain P-53. MINO resistance phenotype of a recombinant thus obtained was transferred by a different mating system to the recipient strain Jucho, once again. Ribosomal proteins of each of the donors, recipients and recombinants were analyzed and compared on 2-dimensional (2D) electrophoresis. Approximately 50 ribosomal proteins were observed in 70S ribosomes. Some proteins were differently electrophoresed in different strains. The 30S ribosomal subunits contained at least 19 proteins and 50S ribosomal subunits contained at least 23 proteins. Some proteins were easily washed off during dissociation of subunits in sucrose gradients. At least one protein (designated F) in both subunits was observed at the same position. One protein designated C in 30S subunits could be co-transferred to the recipient cells together with resistance phenotype at the frequency of 100% in the 30 recombinants examined so far. The other protein designated D in 30S subunits could be transferred at the frequency of 86-88%. Three other proteins in 50S subunits could be co-transferred to the recipient strain at a lower frequency. Minocycline resistance, therefore, could be mapped close to genes encoding the structure of ribosomal proteins in M. smegmatis.     

Binding of chloramphenicol and a fragment of aminoacyl-transfer ribonucleic acid to ribosomes and a ribosome precursor from a mutant of Escherichia coli.

During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.     

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.     

Analysis of Ribosomes from Viomycin-Sensitive and -Resistant Strains of Mycobacterium smegmatis

Viomycin-resistant strains were isolated from Mycobacterium smegmatis. Ribosomes were isolated and tested for drug resistance in subcellular systems containing poly(U) as messenger ribonucleic acid. Resistance to viomycin in these strains was due to altered ribosomes. Further analysis showed that viomycin resistance of two mutants with low level resistance (20 mug/ml) was due to altered 30S ribosomal subunits. Another mutant that was highly resistant to viomycin (1 mg/ml), however, had altered 50S ribosomal subunits.     

Separation of mutant and wild-type ribosomes based on differences in their anti Shine-Dalgarno sequence.

We describe a system to isolate 30S ribosomal subunits which contain targeted mutations in their 16S rRNA. The mutations of interest should be present in so-called specialized 30S subunits which have an anti-Shine-Dalgarno sequence that is altered from 5' ACCUCC to 5' ACACAC. These plasmid-encoded specialized 30S subunits are separated from their chromosomally encoded wild-type counterparts by affinity chromatography that exploits the different Shine-Dalgarno complementarity. An oligonucleotide complementary to the 3' end of wild-type 16S rRNA and attached to a solid phase matrix retains the wild-type 30S subunits. The flow-through of the column contains close to 100% mutant 30S subunits. Toeprinting assays demonstrate that affinity column treatment does not cause significant loss of activity of the specialized particles in initiation complex formation, whereas elongation capacity as determined by poly(Phe) synthesis is only slightly decreased. The method described offers an advantage over total reconstitution from in vitro transcribed mutant 16S rRNA since our 30S subunits contain the naturally occurring base modifications in their 16S rRNA.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation

The 30S ribosomal subunits derived from Escherichia coli TA114, a a temperature-sensitive mutant lacking ribosomal protein S20, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminocyl-tRNAs and templates, including the formation of 30S initiation complexes with fMet-tRNA and mRNA). The 30S (-S20) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site.     

A time-resolved investigation of ribosomal subunit association

The notion that the ribosome is dynamic has been supported by various biochemical techniques, as well as by differences observed in high-resolution structures of ribosomal complexes frozen in various functional states. Yet, the mechanisms and extent of rRNA dynamics are still largely unknown. We have used a novel, fast chemical-modification technique to provide time-resolved details of 16 S rRNA structural changes that occur as bridges are formed between the ribosomal subunits as they associate. Association of different 16 S rRNA regions was found to be a sequential, multi-step process involving conformational rearrangements within the 30 S subunit. Our results suggest that key regions of 16 S rRNA, necessary for decoding and tRNA A-site binding, are structurally altered in a time-dependent manner by association with the 50 S ribosomal subunits.     

Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20

A spontaneous mutant of Escherichia coli K-12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42° C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m⁵C and m⁶₂A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m²G which is not affected at all and m³U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association.     

30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15

Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.     

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.