Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.     

Pleiotropic effects resulting from mutations in genes for ribosomal proteins: analysis of revertants from streptomycin dependence

In order to study the functions of the individual ribosomal proteins and their interaction, a group of revertants from streptomycin dependence to independence was analyzed. Reversion from dependence resulted from a number of different mutational events, all resulting in altered ribosome function. The mutants selected for study exhibited extensive pleiotropy—in addition to the elimination of the requirement for streptomycin for growth, the strains differed from the dependent parent and each other in growth rate, level of streptomycin resistance, effect of antibiotics on viability, rate of subunit assembly in vivo, affinity of isolated ribosomes for streptomycin and functionality of ribosomes in various cell-free assays.There appear to be strong correlations between the level of resistance to streptomycin in growing cells and the ability of the isolated ribosomes to bind streptomycin, the effect of antibiotic on cell-free protein synthesis programmed with natural message (but not poly(U)) and the degree of translational fidelity. There seems to be no relation between level of antibiotic resistance and the overall growth rate, the presence of a defect in ribosome assembly or the ribosomal protein altered by the mutation. Mutations in genes for 30 S proteins S4 and S5 can result in the same phenotype, while different changes in S4 in otherwise isogenic strains result in widely varying phenotypes.The wide variety of effects resulting from single mutational events suggests that each of these changes in a ribosomal protein changes the conformation of the ribosome or its ability to undergo configurational changes.     

Aurintriboxylic acid as both a stimulator and an inhibitor of cell-free protein synthesis by rat heart polyribosomes

Aurintricarboxylic acid is shown to be a powerful stimulator at lower concentrations and a strong inhibitor at higher concentrations of protein synthesis in a heart ribosomes brain supernatant cell free system. The stimulation appears to arise from the prevention of low affinity abortive complex formation.     

Interaction of a fluorescent streptomycin derivative with Escherichia coli ribosomes.

The high salt wash of rabbit reticulocyte ribosomes contains two separate factors which can partially reverse the inhibition of polypeptide chain initiation that results when reticulocyte lysate is incubated in the absence of hemin. These two factors, termed initiation factor (IF) 1 and IF-2, have been separated from each other by chromatography on diethylaminoethyl cellulose and then further purified on hydroxyapatite. IF-1 forms a GTP-dependent complex with methionyl-tRNA_f that is retained on Millipore filters. When these factors are added to a system containing reconstituted, salt-extracted ribosomes, IF-1 promotes the binding of methionyl-tRNA_f to the 40 S subunit, whereas IF-2 promotes the formation of 80 S initiation complexes from 40 S complexes. Addition of small amounts of one factor and a saturating level of the other to the unfractionated lysate and incubation in the absence of hemin produce an additive stimulation of protein synthesis. Each factor can also partially reverse the inhibitory effect of the hemin-controlled translational repressor. The implication of these findings for the mechanism of hemin control of protein synthesis in reticulocyte lysates is discussed.     

Dissection of the mechanism for the stringent factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.     

Morphological stages of Bacillus subtilis sporulation and resistance to fusidic acid.

During spore development of Bacillus subtilis both protein synthesis and sporulation become resistant to the antibiotic fusidic acid. This resistance develops at the time when asymmetric prespore septa are formed. Simultaneously ribosomes lose their ability to bind fusidic acid, as demonstrated by their affinity chromatography with the immobilized drug. Mutants resistant to fusidic acid during growth are oligosporogenous; their sporulation development is blocked before septum formation. These results indicate that normal ribosomes are needed for prespore septation sporulation; only after septation can protein synthesis be maintained, throughout the development period, by fusidate resistant ribosomes.     

Functional interactions of an Escherichia coli ribosomal ATPase.

The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.     

Kinetic studies of streptomycin uptake implicated in self-resistance in a streptomycin producer

Summary Streptomycin (SM)-producingStreptomyces griseus was permeable to extracellular SM during exponential growth, and less permeable during the stationary phase when antibiotic production was maximal. Uptake of [³H] dihydrostreptomycin ([³H]DSM) by the producer organism was abolished by inhibitors of electron transport, sulfhydryl reagents and an uncoupler of oxidative phosphorylation, and it was competitively inhibited by spermidine. These results indicate that SM was taken up by an active transport process via a polyamine transport system. A mutant with lower SM-resistance showed the same level of SM 6-phosphotransferase as the parent strain. It is suggested that selfresistance in the SM-producers is at least partly determined by transport and permeability mechanisms.     

Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein

An 11S protein composed of six polypeptide chains was previously purified from a salt extract of dog pancreas microsomal membranes and shown to be required for translocation of nascent secretory protein across the microsomal membrane (Wistar and Blobel 1980 Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). This 11S protein, termed signal recognition protein (SRP), has been shown here (a) to inhibit translation in the wheat germ cell-free system selectively of mRNA for secretory protein (bovine preprolactin) but not of mRNA for cytoplasmic protein (alpha and beta chain of rabbit globin); (b) to bind with relatively low affinity (apparent KD less than 5 x 10(-5)) to monomeric wheat germ ribosomes; and (c) to bind selectively and with 6,000-fold higher affinity (apparent KD less than 8 x 10(-9)) to wheat germ ribosomes engaged in the synthesis of secretory protein but not to those engaged in the synthesis of cytoplasmic protein. Low- and high- affinity binding as well as the selective translation-inhibitory effect were abolished after modification of SRP by N-ethyl maleimide. High- affinity binding and the selective translation-inhibitory effect of SRP were largely abolished when the leucine (Leu) analogue beta-hydroxy leucine was incorporated into the nascent secretory polypeptide.     

Electron microscope studies on the morphogenesis of plastids

Streptomycin sulphate (2 mg/ml) did not affect the formation of proplastids or the elaboration of prolamellar bodies. The plastids of the streptomycin (SM)-treated cotyledons contained both crystalline prolamellar bodies and ribosomes, and were undistinguishable from the plastids of the water-grown cotyledon. However, plastids from dark-grown SM-treated cotyledons were no longer able to differentiate to more advanced stages of development, even after exposure to light. The plastids of light and dark-grown SM-treated cotyledons often contained prolamellar bodies and abnormal giant grana. Variegation developed in the cotyledons germinated in Hoagland's solution plus SM. The plastids in pale green tissue contained stroma-lamellae and one or two giant grana, whereas in those of pale yellow tissue, many osmiophilic globules, large vacuoles and crystal bodies were observed. It is suggested that the formation of prolamellar bodies may depend on cytoplasmic protein synthesis whereas functional stroma- and grana-lamellae may depend on protein synthesis within the plastids. The inhibitory effects of SM on protein synthesis were used as a tool to test this hypothesis. This work was carried out in the Department of Botany, University of California, Davis, by Grant-GB-11906 from National Science Foundation of U.S.A.     

Chloroplast and Cytoplasmic Ribosomes of Euglena: Selective Binding of Dihydrostreptomycin to Chloroplast Ribosomes

Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The K(diss) for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 x 10(-7) M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm(1) (r)BNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The K(diss) for the chloroplast ribosome-dihydrostreptomycin complex of Sm(1) (r) is 387 x 10(-7) M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 x 10(-7) M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena.     

Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.     

Resistance of bacterial protein synthesis to double-stranded RNA

Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.     

Active center cleft residues of pokeweed antiviral protein mediate its high-affinity binding to the ribosomal protein L3

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP) which catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop (SRL) of the large ribosomal RNA and thereby inhibits the protein synthesis. The ribosomal protein L3, a highly conserved protein located at the peptidyltransferase center of the ribosomes, is involved in binding of PAP to ribosomes and subsequent depurination of the SRL. We have recently discovered that recombinant PAP mutants with alanine substitution of the active center cleft residues (69)NN(70) (FLP-4) and (90)FND(92) (FLP-7) that are not directly involved in the catalytic depurination at the active site exhibit >150-fold reduced ribosome inhibitory activity [(2000) J. Biol. Chem. 275, 3382--3390]. We hypothesized that the partially exposed half of the active site cleft could be the potential docking site for the L3 molecule. Our modeling studies presented herein indicated that PAP residues 90--96, 69--70, and 118--120 potentially interact with L3. Therefore, mutations of these residues were predicted to result in destabilization of interactions with rRNA and lead to a lower binding affinity with L3. In the present structure-function relationship study, coimmunoprecipitation assays with an in vitro synthesized yeast ribosomal protein L3 suggested that these mutant PAP proteins poorly interact with L3. The binding affinities of the mutant PAP proteins for ribosomes and recombinant L3 protein were calculated from rate constants and analysis of binding using surface plasmon resonance biosensor technology. Here, we show that, compared to wild-type PAP, FLP-4/(69)AA(70) and FLP-7/(90)AAA(92) exhibit significantly impaired affinity for ribosomes and L3 protein, which may account for their inability to efficiently inactivate ribosomes. By comparison, recombinant PAP mutants with alanine substitutions of residues (28)KD(29) and (111)SR(112) that are distant from the active center cleft showed normal binding affinity to ribosomes and L3 protein. The single amino acid mutants of PAP with alanine substitution of the active center cleft residues N69 (FLP-20), F90 (FLP-21), N91 (FLP-22), or D92 (FLP-23) also showed reduced ribosome binding as well as reduced L3 binding, further confirming the importance of the active center cleft for the PAP--ribosome and PAP--L3 interactions. The experimental findings presented in this report provide unprecedented evidence that the active center cleft of PAP is important for its in vitro binding to ribosomes via the L3 protein.     

BiP Modulates the Affinity of Its Co-chaperone ERj1 for Ribosomes

Ribosomes synthesizing secretory and membrane proteins are bound to the endoplasmic reticulum (ER) membrane and attach to ribosome-associated membrane proteins such as the Sec61 complex, which forms the protein-conducting channel in the membrane. The ER membrane-resident Hsp40 protein ERj1 was characterized as being able to recruit BiP to ribosomes in solution and to regulate protein synthesis in a BiP-dependent manner. Here, we show that ERj1 and Sec61 are associated with ribosomes at the ER of human cells and that the binding of ERj1 to ribosomes occurs with a binding constant in the picomolar range and is prevented by pretreatment of ribosomes with RNase. However, the affinity of ERj1 for ribosomes dramatically changes upon binding of BiP. This modulation by BiP may be responsible for the dual role of ERj1 at the ribosome, i.e. acting as a recruiting factor for BiP and regulating translation.     

Purification of reticulocyte ribosomes using polyuridylic acid: sepharose chromatography.

Deoxycholate-KCl washed reticulocyte ribosomes are purified by affinity chromatography using Sepharose columns to which polyuridylic acid has been covalently bound. Upon passage through the column under nonenzymic conditions (high Mg²⁺:K⁺ ratio) approximately 10% of the ribosomes are retained. These ribosomes are then eluted with a buffer containing a high K⁺:Mg²⁺ ratio and are assayed for activity in various steps of the elongation process of protein synthesis. Approximately a 3- to 4-fold increase in the various activities compared with control ribosomes is obtained.     

Analysis of the reversible binding of virginiamycin M to ribosome and particle functions after removal of the antibiotic

Type A synergimycins (VM) were shown to act catalytically and to induce two ribosomal alterations: (a) inability to promote polypeptide synthesis; (b) high-affinity binding of type B synergimycins (VS). A claim for irreversible binding of type A synergimycins to ribosomes has promoted the present reinvestigation. Submission of ribosomes from VM-treated bacteria to a purification procedure (supposed to remove the drug, according to a low association constant previously reported) yielded particles still holding residual VM. The formation of VM.ribosome complexes, more stable than previously inferred but without covalent linkage, was deduced from the extractability of complexed VM by organic solvents. Moreover, incubation of these complexes with increasing amounts of anti-VM immunoglobulins progressively restored ribosome activity in protein synthesis. Binding of VS to ribosomes, by fluorimetric titrations in the presence of substoichiometric concentrations of VM, was incompatible with catalytic action of type A synergimycins. Ribosomes from VM-treated bacteria displayed also a higher affinity for VS than did control ribosomes. This property did not disappear when ribosome.VM complexes were incubated with anti-VM IgG, nor when VM-IgG complexes were withdrawn from the reaction mixture by protein A-agarose binding. We can conclude that VM binding produces: (1) an inhibition of ribosome-promoted peptide bond formation, which occurs only in the presence of the drug; and (2) an increase of ribosome affinity for VS, which lasts after VM removal. The linkage of this drug with ribosomes is tight but reversible and its action is stoichiometric.     

Growth-rate-dependent adjustment of ribosome function in the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus was grown at rates between 0.043 and 0.434 doubling/h while maintained as yeasts or at rates between 0.21 and 0.50 doubling/h while maintained as hyphae by altering the composition of the growth medium or the gaseous environment of the cells. Yeasts at the higher growth rates contained many more ribosomes than did yeasts at the lower growth rates. They also had a higher percentage of ribosomes active in protein synthesis and a faster rate of polypeptide-chain elongation than did the slower-growing cells. Hyphal cells at faster growth rates also contained many more ribosomes and showed a faster rate of polypeptide-chain elongation than did slower-growing cells. However, the faster-growing cells had a substantially lower proportion of ribosomes active in protein synthesis than did the slower-growing hyphae. Pulse-chase experiments failed to provide any evidence of protein turnover, which might otherwise invalidate the values calculated for the peptide-chain elongation rates.     

Bovine mitochondrial ribosomes possess a high affinity binding site for guanine nucleotides

Mammalian mitochondrial ribosomes possess a binding site for guanine nucleotides. GTP binds in unit stoichiometry and with high affinity (Kd = 15.3 +/- 2.8 nM) to the small subunit of bovine mitochondrial ribosomes. This binding activity survives high salt washes, indicating that the nucleotide binds to an integral site within this subunit. GDP also binds to the small subunit with high affinity (Kd = 17 +/- 5.8 nm) and in unit stoichiometry. The GTP binding activity can be competed with GDP but not appreciably by other nucleotides, indicating that both GTP and GDP bind specifically and to the same site. The non-hydrolyzable analogs of GTP, guanylyl-5'-imidophosphate, and guanylyl-(beta,gamma-methylene)- diphosphonate also bind to the small subunit, but with reduced affinity. These results indicate that mammalian mitochondrial ribosomes, unlike other ribosomes, are able to interact directly with guanosine triphosphate, suggesting that the bound GTP may be involved in a novel regulatory mechanism in mitochondrial protein synthesis.     

A single mutation in 16S rRNA that affects mRNA binding and translation-termination.

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.