Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

Studies of Ribosomal Diffusion Coefficients Using Laser Light-Scattering Spectroscopy

Using an optical beating technique, the diffusion coefficients and relative scattered intensity of Escherichia coli 70S, 50S, and 30S ribosomes are measured as a function of temperature and Mg²⁺ concentration. For solutions at 10 mM Mg²⁺ and between 0°C and about 40°C, the values of D_20,w obtained are 1.7, 1.9, and ≈2.1 × 10^-7 cm²/s, respectively. Preparative procedures drastically affect these values and equivalent hydrodynamic ellipsoids of revolution models give large axial ratios indicating extensive hydration or a deviation from the assumed shape. Calculations also indicate that the subunits expand upon dissociation. Measurements of D_20,w vs. temperature indicate that 70S particles undergo a conformational change prior to dissociation and can be heat dissociated at 30-32°C at low concentrations. Treatment of 70S ribosomes with EDTA causes a biphasic dissociation reaction. Addition of Mg²⁺ after dissociation with EDTA shows that longer waiting times yield fewer 70S particles and that even short waiting times may yield ribosomes differing from the native conformation. Addition of p-chloromercuribenzoic acid (PCMB) is shown to dissociate 70S particles, but to a lesser extent than ethylenediaminetetraacetic acid (EDTA).     

The initiation codon AUG binds at a hydrophobic site on yeast 40S ribosomal subunits as revealed by fluorescence studies with bis (1,8-anilinonaphthalenesulfonate).

Binding studies of yeast 40S ribosome with bis (1,8-anilinonaphthalenesulfonate) (bis-ANS) revealed the binding of 3-4 molecules of bis-ANS per ribosome with a dissociation constant (Kd) of 1.45 microM. Binding of AUG to the 40S subunits resulted in a concentration-dependent decrease in the bis-ANS fluorescence without displacing all of the bound bis-ANS from the ribosomes. The residual bis-ANS fluorescence at saturation with AUG corresponds to about 3 molecules of bis-ANS per ribosome. Thus AUG displaces one of the bound bis-ANS molecules. The data suggest that AUG binds at a hydrophobic site on the yeast 40S subunit.     

Enzymatic Degradation of Ribosomes During Endogenous Respiration of Pseudomonas aeruginosa.

Gronlund, Audrey F. (University of British Columbia, Vancouver, B.C., Canada), and J. J. R. Campbell. Enzymatic degradation of ribosomes during endogenous respiration of Pseudomonas aeruginosa. J. Bacteriol. 90:1-7. 1965.-From sedimentation analyses it was found that the ribosomal content of Pseudomonas aeruginosa decreased during endogenous respiration. A greater degree of degradation of 50S than 30S ribosomes occurred during the 3-hr starvation period. The enzyme responsible for the initiation of ribosome degradation and present in the ribosome fraction was identified as polynucleotide phosphorylase. The enzyme was inactive in intact 70S ribosomes, but was active in low magnesium ion concentrations which allowed the 70S ribosome to dissociate. Polynucleotide phosphorylase was not solubilized after dissociation of the 70S particle, but remained firmly attached to the 50S and 30S ribosomes, the ribonucleic acid of which served as substrate.     

Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.     

Binding of spectinomycin by ribosomes fromEscherichia coli

The binding of the aminocyclitol antibiotic spectinomycin to 70S ribosomes and to 30S subunits fromEscherichia coli has been investigated. The association was influenced by the presence of messenger RNA. The K_d for [³H]-4 OH-spectinomycin binding to 70S ribosomes was 2×10^–7 M without mRNA (polyinosinic acid), and 1×10^–6 M with polyinosinic acid. Dissociation of the antibiotic from the ribosomes was significantly affected by the presence of a bound messenger RNA, which reduced the rate of dissociation by a factor of 5.7. The presence of mRNA did not influence the association of spectinomycin with the 30S subunit. The dissociation rate from the small subunit was comparable to the rate of dissociation from the 70S ribosome and was not affected by the presence of mRNA.     

Modification of Escherichia coli ribosomes with the fluorescent reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid. Identification of derivatized L31' and studies on its intraribosomal properties

N-[[(Iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS) is a fluorescent reagent which reacts covalently with the free thiol groups of proteins. When the reagent is reacted with the Escherichia coli ribosome under mild conditions, gel electrophoresis shows modification of predominantly two proteins, S18 and L31', which become labeled to an equal extent. When the native (i.e., untreated) ribosome is dissociated into 30S and 50S subunits, only the 30S ribosomal protein S18 reacts with IAEDANS despite the fact that L31' is still present on the large subunit. Upon heat activation of the subunits, a procedure which alters subunit conformation, S18 plus a number of higher molecular weight proteins is modified, but not L31'; the latter reacts with IAEDANS only in the 70S ribosome or when it is free. In contrast to the relatively stable association of L31' with native or with dissociated ribosomes, dissociation of N-[(acetylamino)ethyl]-5-naphthylaminesulfonic acid (AEDANS)-treated ribosomes weakens the AEDANS-L31'/ribosome interaction, resulting, upon gel filtration analysis, in ribosomes devoid of this derivatized protein.     

RatA (YfjG), an Escherichia coli toxin, inhibits 70S ribosome association to block translation initiation

RatA (YfjG) is a toxin encoded by the ratA-ratB (yfjG-yfjF) operon on the Escherichia coli genome. Induction of RatA led to the inhibition of protein synthesis, while DNA and RNA synthesis was not affected. The stability of mRNAs was also unchanged as judged by in vivo primer extension experiments and by Northern blotting analysis. The ribosome profile of the cells overexpressing RatA showed that 70S ribosomes as well as polysomes significantly decreased with concomitant increase of 50S and 30S subunits. The addition of purified RatA to a cell-free system inhibited the formation of 70S ribosomes even in the presence of 6 mM Mg(2+) . RatA was specifically associated with 50S subunits, indicating that it binds to 50S subunits to block its association with 30S subunits leading to the inhibition of formation of 70S ribosomes. However, RatA did not cause dissociation of 70S ribosomes and its anti-association activity was blocked by paromomycin, an inhibitor for IF3, an essential initiation factor, having 21% sequence homology with RatA. Here we demonstrate that RatA is a new E. coli toxin, which effectively blocks the translation initiation step. We propose that this toxin of previously unknown function be renamed as RatA (Ribosome association toxin A).     

Codon-anticodon interaction at the P site is a prerequisite for tRNA interaction with the small ribosomal subunit

The arrival of high resolution crystal structures for the ribosomal subunits opens a new phase of molecular analysis and asks for corresponding analyses of ribosomal function. Here we apply the phosphorothioate technique to dissect tRNA interactions with the ribosome. We demonstrate that a tRNA bound to the P site of non-programmed 70 S ribosomes contacts predominantly the 50 S, as opposed to the 30 S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30 S binding. Protection patterns of tRNAs bound to isolated subunits and programmed 70 S ribosomes were compared. The results suggest the presence of a movable domain in the large ribosomal subunit that carries tRNA and reveal that only approximately 15% of a tRNA, namely residues 30 +/- 1 to 43 +/- 1, contact the 30 S subunit of programmed 70 S ribosomes, whereas the remaining 85% make contact with the 50 S subunit. Identical protection patterns of two distinct elongator tRNAs at the P site were identified as tRNA species-independent phosphate backbone contacts. The sites of protection correlate nicely with the predicted ribosomal-tRNA contacts deduced from a 5.5-A crystal structure of a programmed 70 S ribosome, thus refining which ribosomal components are critical for tRNA fixation at the P site.     

Characterization of ribosomes of a strain ofStreptomyces aureofaciens producing chlortetracycline

These studies were designated to investigate the effect of chlortetracycline on sedimentation properties of polysomes and ribosomes present in the chlortetracycline producing strain ofStreptomyces aureofaciens. In presence of chlortetracycline polysomes and ribosomes are more stable than the bacterial ones. At lower chlortetracycline concentrations (1–5 μg/ml) dissociation of polysomes into 70 S monomers was not observed. Ribosomes in higher concentration of chlortetracycline (400 μg/ml) form aggregates. A decrease of Mg²⁺ to 0.1mm caused dissociation of ribosomes to two subunits and in this state none of indicated concentrations of chlortetracycline caused aggregation. The exact sedimentation values of ribosomes and ribosomal subunits were calculated from extrapolation to infinite dilution. S_20,w for monomer form was 68.8, and for ribosomal subunits 49.8 and 31.2 respectively. Ribosomal RNA sedimentates as two Schlieren peaks of 16 S and 22 S. It was found that 30 S subunits contain 15 structural proteins, while 21 proteins were resolved from 50 S subunits.     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.     

Binding of the antibiotic vernamycin in Balpha to Escherichia coli ribosomes

The interaction of the antibiotic vernamycin Bα with Escherichia coli ribosomes has been studied. The antibiotic is bound to 70S ribosomes and 50S subunits but not to the 30S subunit or to polysomes. The binding of the antibiotic requires K⁺ or NH⁺₄ and Mg²⁺. At saturation approximately 0.5 mole of antibiotic is bound per mole of ribosomes. The vernamycin Bα-ribosome complex is unstable. The bound antibiotic is readily displaced by nonradioactive vernamycin Bα and by a number of other antibiotics which are known to interact with the 50S subunit. The dissociation of the vernamycin Bα-ribosome complex is prevented by the simultaneous binding of vernamycin A. The binding sites for A and Bα are distinguishable since both drugs are able to bind simultaneously and neither prevents binding of the other, Ribosomes isolated from an erythromycin-resistant mutant are incapable of binding vernamycin A and Bα, indicating that the mutated protein responsible for resistance to erythromycin distorts the ribosome making it also unreceptive for the vernamycins.     

Alteration of the structure of theEscherichia coli ribosomes on treatment with Fab fragment of immunoglobulin raised against the ribosomes

Rabbits were immunised againstEscherichia coli ribosomes and the partially purified immunoglobulin G fraction had maximum ability to precipitate the ribosomes as well as the extracted ribosomal proteins. By digestion of immuno-globulin G with papain, monovalent Fab fragments were produced. The 70 S ribosome and its subunits (50 S and 30 S) were separately treated with Fab and then tested in the kinetic assay of degradation of ribosomes by ribonuclease I at various Mg²⁺ concentrations. Treated ribosomes and their subunits were degraded at faster rates than the nontreated ones; the rates in both the control and the treated cases were dependent on the concentration of Mg²⁺. These results indicate the unfolding of the structure of the ribosome on treatment with antibody fragments, which may be due to the weakening of the interaction between rRNAs and ribosomal proteins.     

Dissociation of 70 S E. coli ribosomes induced by a ribosomal factor (DF). Electrophoretic studies of the ribosomal particles

The dissociation of purified 70 S.E. coli ribosomes, induced by the dissociation factor DF, has been studied by submitting the reaction mixtures to electrophoresis on polyacrylamide gels. The electrophoretic analysis of the ribosome mixtures revealed a heterogeneity which escaped detection by conventional sucrose gradient centrifugation. Increasing amounts of DF in the reaction mixtures converted 70 S ribosomes to particles (designated 70 S (I)) which migrate slower in the electric field than the original 70 S ribosomes. These 70 S (I) ribosomes still consist of both subunits. They dissociate upon further raising the DF concentration.     

An initiation factor causing dissociation of E. coli ribosomes

Purification of crude initiation factors, essential for polypeptide synthesis in cell-free systems of E. coli, yielded a fraction DF which causes dissociation of 70 S ribosomes. Its stoichiometric action on 70 S ribosomes is antagonized by increasing Mg(2+) concentrations but not by the addition of 30 S and 50 S subunits washed with high salt concentration. GTP did not stimulate this dissociating action. 2 &mgr;g of our most purified preparation caused 100% dissociation of 100 &mgr;g of 70 S ribosomes without added GTP. DF-induced dissociation is a very rapid process at 37 degrees C and is temperature-dependent in the range of 0 degrees -37 degrees C. DF, which is thermolabile factor, is much less or not effective with complexed 70 S ribosomes bearing peptidyl-tRNA and mRNA.     

Leaderless mRNAs bind 70S ribosomes more strongly than 30S ribosomal subunits in Escherichia coli

By primer extension inhibition assays, 70S ribosomes bound with higher affinity, or stability, than did 30S subunits to leaderless mRNAs containing AUG or GUG start codons. Addition of translation initiation factors affected ribosome binding to leaderless mRNAs. Our results suggest that translation of leaderless mRNAs might initiate through a pathway involving 70S ribosomes or 30S subunits lacking IF3.     

Silver Plated Ribosomes

WE recently reported that dissociation of free 70S ribosomes from E. coli to 30S and 50S subunits could occur during sedimentation in buffer containing K⁺ as the predominant monovalent cation¹. Spermidine at a physiological concentration (1 mM) specifically prevented the dissociation and we suggested that the dissociation that occurred in its absence was due to loss of the polyamine from the 70S ribosome. We have subsequently found that this hypothesis is wrong and that the dissociation that took place in the original experiments was due not primarily to loss of spermidine, but to a reaction with silver present in very small amounts in the AR KCl used in the buffer.     

Study of the surface of Escherichia coli ribosomes and ribosomal particles by the tritium bombardment method

A new technique of atomic tritium bombardment has been used to study the surface topography of Escherichia coli ribosomes and ribosomal subunits. The technique provides for the labeling of proteins exposed on the surface of ribosomal particles, the extent of protein labeling being proportional to the degree of exposure. The following proteins were considerably tritiated in the 70S ribosomes: S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L1, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27. A conclusion is drawn that these proteins are exposed on the ribosome surface to an essentially greater extent than the others. Dissociation of 70S ribosomes into the ribosomal subunits by decreasing Mg2+ concentration does not lead to the exposure of additional ribosomal proteins. This implies that there are no proteins on the contacting surfaces of the subunits. However, if a mixture of subunits has been subjected to centrifugation in a low Mg2+ concentration at high concentrations of a monovalent cation, proteins S3, S5, S7, S14, S18 and L16 are more exposed on the surface of the isolated 30S and 50S subunits than in the subunit mixture or in the 70S ribosomes. The exposure of additional proteins is explained by distortion of the native quaternary structure of ribosomal subunits as a result of the separation procedure. Reassociation of isolated subunits at high Mg2+ concentration results in shielding of proteins S3, S5, S7 and S18 and can be explained by reconstitution of the intact 30S subunit structure.     

A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.