Inhibition of elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes by Shiga-like toxin I (VT1) from Escherichia coli O157:H7 and by Shiga toxin

A Vero toxin (VT1 or Shiga-like toxin I) from Escherichia coli O157:H7 was found to inactivate 60S ribosomal subunits of rabbit reticulocyte resulting in the inhibition of protein synthesis. The mode of inhibition of both VT1 and Shiga toxin was similar and involved the blocking of elongation-1-dependent binding of aminoacyl-tRNA of ribosomes. Non-enzymatic binding of aminoacyl-tRNA to ribosomes, peptide bond formation and translocation were not inhibited by either VT1 or Shiga toxin.     

Increase of degree of spermidine stimulation of polypeptide synthesis in the presence of phosphate

The addition of phosphate caused an increase in the degree of spermidine stimulation of polypeptide synthesis in an Escherichia coli and a wheat germ cell-free system. Optimal stimulation of polypeptide synthesis was observed at 20 mm phosphate for both systems, but concentrations of phosphate up to 40 mm had no additional effect. The increase of degree of spermidine stimulation in the presence of phosphate in an E. coli cell-free system occurred at the level of aminoacyl-tRNA binding to ribosomes and not at the level of peptide bond formation, translocation, or aminoacyl-tRNA formation. From the results of studies on RNase A sensitivity of ribosomal subunits and the effect of antibiotics known to act on the 30 S ribosomal subunits, it is suggested that the nature of the 30 S ribosomal subunits is changed by phosphate so that the degree of spermidine stimulation of polypeptide synthesis is increased.     

Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine. V. Methylation dependence on the assembly of E. coli 50 S ribosomal subunits.

An ethionine-containing submethylated particle related to the 50 S ribosomal subunit has been isolated from Escherichia coli grown in the presence of ethionine. This particle (E-50S) lacks L16, contains reduced amounts of L6, L27, L28 and L30 and possesses a more labile and flexible structure than the normal 50 S subunit. The E-50S particle has defective association properties and is incapable of peptide bond formation. It can be converted to an active 50 S ribosomal subunit when ethionine-treated bacteria are incubated under conditions which permit methylation of submethylated cellular components (presence of methionine) in the absence of de novo protein and RNA synthesis (presence of rifampicin).Total reconstitution of 50 S ribosomal subunits in vitro using normal 23 S and 5 S ribosomal RNA and proteins prepared from E-50S particles yields active subunits only if L16 is also added. The hypothesis that E-50S particles accumulate in ethionine-treated bacteria because the absence of methylation of one or more of their components blocks a late stage (L16 integration) in the normal 50 S assembly process is discussed.     

Archaebacterial and eukaryotic ribosomal subunits can form active hybrid ribosomes

Purified ribosomal subunits from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus are able to recognize ribosomal subunits from the yeast Saccharomyces cerevisiae forming hybrid monosomes that can be revealed by sucrose gradient analysis and are active in peptide bond formation. Both reciprocal combinations (archaebacterial 30 S + eukaryotic 60 S and archaebacterial 50 S + eukaryotic 40 S) are functional. In contrast, no hybrid couples are formed between subunits of yeast and Escherichia coli ribosomes. These results indicate that ribosomes of at least one archaebacterial species share specific structural features with those of the lower eukaryote S. cerevisiae.     

Biochemical evidence of translational infidelity and decreased peptidyltransferase activity by a sarcin/ricin domain mutation of yeast 25S rRNA

A C→U mutation (rdn5) in the conserved sarcin/ricin domain of yeast 25S rRNA has been shown to cause translational suppression and paromomycin resistance. It also separates the killing from the misreading effect of this antibiotic. We confirm these findings and provide in vitro evidence that rdn5 causes a 3-fold increase in translational errors and resistance to paromomycin. The role of this 25S rRNA domain in ribosome's decoding function was further demonstrated when 60S subunits from rdn5 cells were combined with 40S subunits from cells carrying an error-prone mutation in the eukaryotic accuracy center ribosomal protein S23, an homologue of Escherichia coli S12. These hybrids exhibited an error frequency similar to that of rdn5 alone, despite the error-prone mutation in S23. This was accompanied by extreme resistance to paromomycin, unlike the effects of the individual mutations. Furthermore, rdn5 lowers peptidyltransferase activity measured as a second-order rate constant (k_cat/K_s) corresponding to the rate of peptide bond formation. This mutation was also found to affect translocation. Elongation factor 2 (EF2)-dependent translocation of Ac-Phe-tRNA from the A- to P-site was achieved at an EF2 concentration 3.5 times lower than in wild type. In conclusion, the sarcin/ricin domain of 25S rRNA influences decoding, peptide bond formation and translocation.     

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.     

Biological regulation through protein disulfide bond cleavage

Abstract

It is thought that disulfide bonds in secreted proteins are inert because of the oxidizing nature of the extracellular milieu. We have suggested that this is not necessarily the case and that certain secreted proteins contain one or more disulfide bonds that can be cleaved and that this cleavage is central to the protein's function. This review discusses disulfide bond cleavage in the secreted soluble protein, plasmin. Cleavage of plasmin disulfide bond(s) triggers peptide bond cleavage and formation of the tumour angiogenesis inhibitor, angiostatin. Tumour cells secrete phosphoglycerate kinase which facilitates cleavage of the plasmin disulfide bond(s). Phosphoglycerate kinase is not a conventional disulfide bond reductase. We propose that phosphoglycerate kinase facilitates cleavage of a particular plasmin disulfide bond by hydroxide ion, which results in formation of a sulfenic acid and a free thiol. The free thiol is then available to exchange with another nearby disulfide bond resulting in formation of a new disulfide and a new free thiol. The reduced plasmin is then susceptible to discreet proteolysis which results in release of angiostatin.     

Both ends ofEscherichia coli ribosomal protein S13 are immunochemically accessiblein situ

To investigate the structure ofEscherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopesin situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13in situ.     

Rapid peptide bond formation on isolated 50S ribosomal subunits

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.     

Assembly in vivo of mouse muscle acetylcholine receptor: identification of an alpha subunit species that may be an assembly intermediate

We have studied assembly of acetylcholine receptor in vivo using subunit-specific monoclonal antibodies and immunoprecipitation with alpha-bungarotoxin and antitoxin. We have identified three distinct forms of the alpha subunit. The newly synthesized alpha subunit species has a sedimentation coefficient of 5S and is recognized only by antibody specific for SDS-denatured alpha subunit. We have called this species alpha 61. The 5S alpha Tx species is not associated with beta subunits and is probably monomeric. alpha Tx is formed from alpha 61 with a half-time of 15 min and an efficiency of approximately equal to 30%. Formation of alpha Tx involves a conformational change, and we suggest that this conformation is dependent upon or stabilized by disulfide bond formation. The assembly of alpha Tx with beta subunits (and probably gamma and delta) into a 9S complex appears to be an efficient but slow process requiring more than 90 min. Unassembled alpha 61 subunits are degraded rapidly. However, subunit degradation is a result of failure to assemble, rather than its cause.     

The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit

The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.     

Reaction of ribosomal sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid)

The number of sulfhydryl groups in the Escherichia coli ribosome has been measured by titration with 5,5′-dithiobis(2-nitrobenzoic acid). Under denaturing conditions, there are 38.8 ± 1.0 titratable thiols per 70 S ribosome and 22.8 ± 0.3 and 12.9 ± 0.3 titratable thiols per 50 S and 30 S subunits, respectively. Three categories of thiol groups can be distinguished in the native 70 S ribosome, a “fast reacting” class of about 3 residues, a “slow reacting” class of about 10 residues and a “buried” class including about 26 residues. The addition of polyuridylic acid to reaction mixtures protects a fast-reacting thiol in the 30 S subunit belonging to protein S1.The addition of urea to ribosome solutions makes the buried residues titratable. Denaturation occurs as a sharp transition at a urea concentration between 4 and 4.5 m. Urea does not fully dissociate the ribosome into RNA and protein. Instead, in the case of the 30 S subunit, a slowly sedimenting particle forms in the presence of urea, containing roughly 65% of the normal amount of protein.     

Engineering a disulfide bond and free thiols in the lantibiotic nisin Z.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.     

Nonequivalence of the two subunits of horse erythrocyte glutathione transferase in their reaction with sulfhydryl reagents

Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activity. A peculiar feature of these protein thiol groups is that they react nonidentically with a number of thiol blocking reagents, i.e. iodacetamide, bromopyruvate, N-ethylmaleimide, and 1-chloro-2,4-dinitrobenzene. Also many disulfides react with one thiol group 5- to 10-fold more rapidly than with the other. The two mixed disulfides so formed also have different rates of reactivation by dithiothreitol. All the structural and kinetic data reported in this paper indicate a nonsymmetrical association of two identical subunits, or alternatively heterodimeric structure with subunits of very similar charge and size.     

SPARK--a novel method to monitor ribosomal peptidyl transferase activity

The key enzymatic activity of the ribosome is catalysis of peptide bond formation. This reaction is a target for many clinically important antibiotics. However, the molecular mechanisms of the peptidyl transfer reaction, the catalytic contribution of the ribosome, and the mechanisms of antibiotic action are still poorly understood. Here we describe a novel, simple, convenient, and sensitive method for monitoring peptidyl transferase activity (SPARK). In this method, the ribosomal peptidyl transferase forms a peptide bond between two ligands, one of which is tritiated whereas the other is biotin-tagged. Transpeptidation results in covalent attachment of the biotin moiety to a tritiated compound. The amount of the reaction product is then directly quantified using the scintillation proximity assay technology: binding of the tritiated radioligand to the commercially available streptavidin-coated beads causes excitation of the bead-embedded scintillant, resulting in detection of radioactivity. The reaction is readily inhibited by known antibiotics, inhibitors of peptide bond formation. The method we developed is amenable to simple automation which makes it useful for screening for new antibiotics. The method is useful for different types of ribosomal research. Using this method, we investigated the effect of mutations at a universally conserved nucleotide of the active site of 23S rRNA, A2602 (Escherichia coli numbering), on the peptidyl transferase activity of the ribosome. The activities of the in vitro reconstituted mutant subunits, though somewhat reduced, were comparable with those of the subunits assembled with the wild-type 23S rRNA, indicating that A2602 mutations do not abolish the ability of the ribosome to catalyze peptide bond formation. Similar results were obtained with double mutants carrying mutations at A2602 and another universally conserved nucleotide in the peptidyl transferase center, A2451. The obtained results agree with our previous conclusion that the ribosome accelerates peptide bond formation primarily through entropic rather than chemical catalysis.     

Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits.

Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, M_r 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented.     

Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes

Protein synthesis in the ribosome's large subunit occurs within an active site comprised exclusively of RNA. Mutational studies of rRNA active site residues could provide valuable insight into the mechanism of peptide bond formation, but many of these mutations cause a dominant lethal phenotype, which prevents production of the homogeneous mutant ribosomes needed for analysis. We report a general method to affinity purify in vivo assembled 50S ribosomal subunits containing lethal active site mutations via a U1A protein-binding tag inserted onto the 23S rRNA. The expected pH-dependent formation of the A2450+C2063 wobble pair has made it a potential candidate for the pH-dependent conformational change that occurs within the ribosomal active site. Using this approach, the active site A2450+C2063 pair was mutated to the isosteric, but pH-independent, G2450•U2063 wobble pair, and 50S subunits containing the mutations were affinity purified. The G•U mutation caused the adjacent A2451 to become hyper-reactive to dimethylsulfate (DMS) modification in a pH-independent manner. Furthermore, the G•U mutation decreased both the rate of peptide bond formation and the affinity of the post-translocation complex for puromycin. The reaction rate (k_pep) was reduced ~200-fold for both puromycin and the natural aminoacyl-tRNA A-site substrate. The mutations also substantially altered the pH dependence of the reaction. Mutation of this base pair has significant deleterious effects upon peptidyl transferase activity, but because G•U mutation disrupts several tertiary contacts with the wobble pair, the assignment of A2450 as the active site residue with the neutral pK_a important for the peptidyl transferase reaction cannot be fully supported or excluded based upon these data.     

Association of nascent polypeptide with 30S ribosomal subunits

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg²⁺ concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).     

Functional epitopes at the ribosome subunit interface

The ribosome is a 2.5-MDa molecular machine that synthesizes cellular proteins encoded in mRNAs. The 30S and 50S subunits of the ribosome associate through structurally defined intersubunit bridges burying 6,000 A(2), 80% of which is buried in conserved RNA-RNA interactions. Intersubunit bridges bind translation factors, may coordinate peptide bond formation and translocation and may be actively remodeled in the post-termination complex, but the functional importance of numerous 30S bridge nucleotides had been unknown. We carried out large-scale combinatorial mutagenesis and in vivo selections on 30S nucleotides that form RNA-RNA intersubunit bridges in the Escherichia coli ribosome. We determined the covariation and functional importance of bridge nucleotides, allowing comparison of the structural interface and phylogenetic data to the functional epitope. Our results reveal how information for ribosome function is partitioned across bridges, and suggest a subset of nucleotides that may have measurable effects on individual steps of the translational cycle.