Crosslinking of chemically reactive A-U-G analogs to ribosomal proteins within initiation complexes.

A-U-G analogs, either reactive on their 5′ or their 3′ side, were employed in affinity labeling of the ribosomal A-U-G binding site. These experiments have been carried out such that the chemically reactive A-U-G analog became covalently bonded to ribosomal proteins only in the presence of fMet-tRNA_f^Met and initiation factors. Subsequent radioimmunodiffusion of A-U-G-labeled proteins identified proteins IF3, S1, S18, S21 and L11 as being in the neighborhood of the ribosomal codon binding site. A location of reactive sites of these proteins relative to the P or A site bound codon is, however, not clear.The A-U-G labeling results are quantitatively as well as qualitatively very different in the absence or presence of fMet-tRNA_f^Met. It is concluded, therefore, that fMet-tRNA_f^Met directs A-U-G into its final binding site. Streptomycin cannot release fMet-tRNA_f^Met from initiation complexes which contain irreversibly bound 5′- {4-(bromoacetamido)phenylphospho}-adenylyl-(3′–5′)-uridylyl-(3′–5′)-guanosine. This suggests that codon-anticodon interaction between A-U-G and fMet-tRNA_f^Met is still intact in the P site of the ribosome.     

The effect of removal or replacement with proflavine of the Y base in the anticodon loop of yeast tRNAPhe on binding into the acceptor or donor sites of reticulocyte ribosomes

Phe-tRNA from yeast has a highly modified nucleoside, called Y, adjacent to the 3′ side of its anticodon, that can be removed or replaced with proflavine. In a protein-synthesizing system from rabbit reticulocytes, poly (U)-directed binding and polyphenylalanine synthesis are low with these modified Phe-tRNA species relative to the corresponding values with unmodified Phe-tRNA. However, polymerization can be increased with relatively large amounts of elongation factor I. The modified Phe-tRNA species bound to the ribosomes with poly(U) either in the presence or absence of elongation factor I and GTP is immediately reactive in the peptidyl transferase reaction measured by the formation of diphenylalanine or phenylalanyl-puromycin. It appears to have been bound directly into the donor ribosomal site by either the nonenzymatic mechanism involving Mg²⁺ or by the enzymatic mechanism involving EF-I and GTP.     

Location of protein S1 of Escherichia coli ribosomes at the ''A''-site of the codon binding site. Affinity labeling studies with a 3''-modified A-U-G analog.

An affinity analog with a 5-bromoacetamido uridine 5'-phosphate moiety bonded to the 3' end of A-U-G has been prepared with the aid of polynucleotide phosphorylase. This 3'-modified, chemically reactive A-U-G analog was used to probe the ribosomal codon binding site. The yield of the reaction depended strongly on the ribosomal source and was sensitive to salt-washing ribosomes. The major crosslinking product was identified to be protein S1. Since the reaction of this 3'-modified A-U-G programmed ribosomes for Met-tRNA-Met-M binding, it is concluded that protein S1 is located at or near the 3'-side of the ribosomal codon binding site.     

Stimulation of enzymatic phe-tRNA binding to mammalian ribosomes by thallium ions at concentrations blocking other ribosomal functions.

Thallium acetate (TIOAc) effectively stimulates poly(U)-directed Phe-tRNA binding to mouse ascitic tumour ribosomes under conditions when other ribosomal functions are completely blocked. The TI+ optimum is about 200 mM. The reaction is stimulated by EF-1, but not significantly by GTP. EF-1-dependent ribosomal GTPase is inhibited by T1+. The isolated Phe-tRNA . ribosome complex is relatively stable. The bound Phe-tRNA does not react with puromycin in the presence of 175 mM KCl. The complex formed in the presence of 90-100 mM TlOAc can, after isolation, be directly utilized for polyphenylalanine synthesis. The complex formed at 200 mM TlOAc is less active, apparently because of damage to the 60-S subunits. TlOAc at low concentrations (8 mM) stimulates K+ -containing poly(U)-translating systems, probably by stabilizing the translation complex.     

tRNA binding capacities of ribosomal subunits from the archaebacterium Halobacterium halobium

tRNA saturation experiments were performed with ribosomal subunits from the extreme halophilic archaebacterium Halobacterium halobium. In the presence of poly(U) the 30S subunit could bind equally well one AcPhe-tRNAPhe, Phe-tRNAPhe, or deacylated tRNAPhe molecule, respectively. Binding experiments with a mixture of two differently labeled tRNA species revealed that all three kinds of tRNA bound to one and the same binding site on the 30S subunit. Poly(U) dependent binding to the 50S subunit was insignificant for AcPhe-tRNA and Phe-tRNA. In the absence of poly(U) both AcPhe-tRNAPhe and Phe-tRNAPhe showed no significant binding to either subunit, whereas the binding of deacylated tRNAPhe could not be clearly determined. These results are in good agreement with those obtained from ribosomal subunits of the eubacterium Escherichia coli.     

Puromycin reaction of the A-site bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.     

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.     

Comparison of the reactions of chemically reactive analogs of U-G-A and of A-U-G with ribosomes of Escherichia coli.

The chemically reactive analog of U-G-A, 5'-(4-(Bromo-[2-14C] acetamido) phenylphospho) - uridylyl-(3'-5') - guanylyl-(3'-5') adenosine has a 20 fold lower affinity to 70S ribosomes than the corresponding analog of A-U-G though the U-G-A analog also preferentially reacts with protein S18 of 70S ribosomes. This reaction programs ribosomes for EF-T dependent Trp-tRNATrp-suIII binding. Therefore, it is concluded that this protein is part of the A'-site of the ribosomal codon binding site. Reaction of the U-G-A analog with 30S subunits lead to a predominant crosslinking of U-G-A to proteins S4 and S18. In contrast, a comparable reaction of the A-U-G analog with 30S subunits lead to a predominant crosslinking of A-U-G to proteins S4 and S12 (Pongs, O., Stoffler, G.A., Lanka, E., (1975) J. Mol. Biol. 99, 301). Since protein S12 is located at the 'P' site of the ribosomal codon binding site, it is proposed that the U-G-A analog does not bind at this site.     

Nuclease digestion patterns as a criterion for nucleosome orientation in the higher order structure of chromatin

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.     

Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.     

The translocation inhibitor tuberactinomycin binds to nucleic acids and blocks the in vitro assembly of 50S subunits.

Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.     

Identification of cysteine-10 of protein S18 as part of the mRNA-binding site of Escherichia coli ribosomes by affinity-labeling studies with a chemically reactive A-U-G analog.

The reaction of a bromoacetamidophenyl derivative of the initiation codon A-U-G (A-U-G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A-U-G-directed fMet-tRNAfMet binding into the puromycin-sensitive site of ribosomes and stimulates elongation-factor-dependent binding of Met-tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl-tRNA binding site of E. coli ribosomes. Peptide as well as amino acid analysis shows that the reaction between A-U-G and ribosomes took place at cysteine-10 of protein S18. A-U-G could not be crosslinked to ribosomal proteins of the temperature-sensitive E. coli strain 258ts, where arginine-11 of protein S18 is replaced by a cysteine residue.     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Enzymatic binding of aminoacyl transfer ribonucleic acid to ribosomes: the study of binding sites of 2' and 3' isomers of aminoacyl transfer ribonucleic acid.

The mechanism of enzymatic binding of AAtRNA to the acceptor site Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A-(2'-Phe)H, and C-A(2'H)Phe. T-psi-C-Gp was used as a model of loop IV of tRNA. The EF-T dependent binding of Phe-tRNA to ribosomes (in the presence of either GTP or GMPPCP) and the GTPase activity associated with EF-T dependent binding of the Phe-tRNA were inhibited by C-A-Phe,C-A(2'Phe)H, and C-A(2'H)Phe. These aminoacyl oligonucleotides inhibit both the formation of ternary complex EF-Tu-GTP-AA-tRNA and the interaction of this complex with the ribosomal A site. The uncoupled EF-Tu dependent GTPase (in the absence of AA-tRNA) was also inhibited by C-A-Phe, C-A(2'Phe)H, and C-A(2'H)Phe, while nonenzymatic binding of Phe-tRNA to the ribosomal A site was inhibited by C-A-Phe and C-A(2'-Phe)H, but not by C-A(2'H)Phe. The tetranucleotide T-psi-C-Gp inhibited both enzyme binding of Phe-tRNA and EF-T dependent GTP hydrolysis. However, inhibition of the latter reaction occured at a lower concentration of T-psi-C-Gp suggesting a specific role of T-psi-C-Gp loop of AA-tRNA in the GTPase reaction. The role of the 2' and 3' isomers of AA-tRNA during enzymatic binding to ribosomes is discussed and it is suggested that 2' leads to 3' transacylation in AA-tRNA is a step which follows GTP hydrolysis but precedes peptide bond formation.     

Apparent association constants of tRNAs for the ribosomal A, P, and E sites.

Association constants for tRNA binding to poly(U) programmed ribosomes were assessed under standardized conditions with a single preparation of ribosomes, tRNAs, and elongation factors, respectively, at 15 and 10 mM Mg2+. Association constants were determined by Scatchard plot analysis (the constants are given in units of [10(7)/M] measured at 15 mM Mg2+): the ternary complex Phe-tRNA.elongation factor EF-Tu.GTP (12 +/- 3), Phe-tRNA (1 +/- 0.4), AcPhe-tRNA (0.7 +/- 0.3), and deacylated tRNA(Phe) (0.4 +/- 0.15) bind with decreasing affinity to the A site of poly(U)-programmed ribosomes. tRNA(Phe) (7.2 +/- 0.8) binds to the P site with higher affinity than AcPhe-tRNA (3.7 +/- 1.3). The affinity of the E site for deacylated tRNA(Phe) (1 +/- 0.2) is about the same as that of the A site for AcPhe-tRNA (0.7 +/- 0.3). At lower Mg2+ concentrations the affinity of the E site ligand becomes stronger relative to the affinities of the A site ligands. Phe-tRNA and ternary complexes can occupy the A site at 0 degrees C in the presence of poly(U) even if the P site is free, whereas, as already known, deacylated tRNA or AcPhe-tRNA bind first to the P site of programmed ribosomes. Hill plot analyses of the binding data confirm an allosteric linkage between A and E sites in the sense of a negative cooperativity.     

Viomycin favours the formation of 70S ribosome couples

Summary The peptide antibiotic viomycin at a concentration of 10 M inhibits E. coli ribosomes to the extent of about 70% as measured in the poly(U) system, and to about 85% in a natural mRNA (R17) system. Ribosomes from M. smegmatis show no activity at all at this concentration of the antibiotic. Experiments on the Mg²⁺ dependent dissociation and association of the ribosomal subunits revealed that viomycin stabilizes the 70S couples and promotes association of ribosomal subunits. This response is related to the drug action as indicated by the observation that viomycin resistant strains are not affected by viomycin with respect to dissociation and 70S couple information. A model for the inhibitory action of the drug is proposed.     

Crosslinking of phenylalanyl-tRNA to the ribosomal A site via a photoaffinity probe attached to the 4-thiouridine residue is exclusively to ribosomal protein S19

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, can be crosslinked to 30 S ribosomal protein when the tRNA is placed at the ribosomal A site. This protein has now been identified by immunological methods. The protein-[3H]Phe-tRNA covalent complex, obtained by extraction with 6 M-urea, was reacted separately with each of the 21 purified antisera to 30 S ribosomal proteins. The double antibody technique was used. Anti-S19 was the only antiserum able to precipitate the radioactivity, and 66 to 81% of the added radioactivity could be precipitated. The same result was obtained with three different ribosome preparations, at low as well as high crosslinking yield, with dipeptidyl-tRNA in the A site as well as aminoacyl-tRNA, and when binding and crosslinking were performed at 20 mM-Mg2+ instead of at 5 mM. Therefore, when aminoacyl-tRNA or peptidyl-tRNA is in the ribosomal A site, position 8, which is always uridine or 4-thiouridine, must be within 9 A of protein S19.     

Aminoacyl-tRNA-elongation factor Tu-ribosome interaction leading to hydrolysis of guanosine 5'-triphosphate

We investigated the elongation factor Tu (EF-Tu) dependent binding of Phe-tRNA and Phe-tRNAs with the nicks at positions 46, 37, and 17 to the Escherichia coli 70S ribosome-poly(U)-tRNAPhe complex. Binding of Phe-tRNA1-45 + 47-76, Phe-tRNA1-36 + 38-76, or Phe-tRNA1-16 + 17-76 to the 70S ribosome has been found to be poly(U) X tRNA dependent and, similar to that of intact Phe-tRNA, is inhibited by the antibiotic thiostrepton. We have further found that, contrary to a previous report [Modolell, J., Cabrer, B., Parmeggiani, A., & Vazquez, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1796], the EF-Tu-ribosome GTPase mediated by Phe-tRNA is not inhibited by thiostrepton; rather, the drug stimulates the endogenous GTPase of the EF-Tu X 70S ribosome. Phe-tRNA fragments 47-76, 38-76, and 17-76 all promote the EF-Tu X GTPase reaction in the presence of 70S ribosome-poly(U)-tRNAPhe yeast. Moreover, since the GTPase-promoting activities of both the short and long fragments are similar, it appears that the most important aminoacyl transfer ribonucleic acid (aa-tRNA) interaction with EF-Tu occurs alongside its 3' quarter. Thiostrepton slightly stimulates the GTPase activity of these Phe-tRNA fragments. Although the Phe-tRNA1-36 + 38-76 cannot bind to poly(U) during its binding to 70S ribosomes, its binding at high Mg2+ concentration occurs at the A site. Thus, most of the bound modified Phe-tRNA functions as the acceptor in the peptidyltransferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The action of virginiamycin M on the acceptor, donor, and catalytic sites of peptidyltransferase

Virginiamycin M inhibits both peptide bond formation and binding of aminoacyl-tRNA to bacterial ribosomes, and induces a lasting inactivation of the 50 S subunit (50 S). In the present work, the effects of this antibiotic on the acceptor and donor sites of peptidyltransferase have been explored, in the presence of virginiamycin M as well as after its removal. Virginiamycin M inhibited the binding of puromycin to ribosomes and reduced both the enzymatic and nonenzymatic binding of Phe-tRNA to the A site by inducing its release from the ribosomes (similar effects were observed with 50 S), whereas the antibiotic had no effect on the binding of unacylated tRNAPhe to the same site. Moreover, virginiamycin M caused Ac-Phe-tRNA or Phe-tRNA to be released from the ribosomal P site, when complexes were incubated with unacylated tRNA, elongation factor G, and GTP (similar finding with 50 S). Instead, peptide bond formation between Ac-Phe-tRNA positioned at the P site and Phe-tRNA at the A site was found to take place, albeit at a very low rate, in the presence of the antibiotic. The overall conclusion is that both the acceptor and donor substrate binding sites of the peptidyltransferase, which interact with the aminoacyl moiety of tRNA, are permanently altered upon transient contact of ribosomes with virginiamycin M.     

Effect of magnesium ions on the tertiary structure of the hepatitis C virus IRES and its affinity for the cyclic peptide antibiotic viomycin

A key ion-dependent folding unit within the hepatitis C IRES comprises the IIIef junction and pseudoknot. This region is also important in recruitment of the 40S ribosomal subunit. Here, circular dichroism is used to study the influence of metal ions on the structure and stability of this region. Comparison of the thermal stability of an IRES fragment encompassing subdomains IIIe/f and IV (named 3EF4) with that of a larger fragment also possessing subdomain IIId (3DEF4) indicates an additional stabilizing effect of Mg(2+) ions on the latter fragment. Magnesium and potassium ions stabilize both fragments through nonspecific counterion effects. The additional effect of magnesium on 3DEF4, observed in the absence or presence of 100 mM KCl, is attributed to a nonspecific but high-affinity site for metal ions created by a region of unusual high charge density. Subdomain IIId presumably participates in tertiary packing interactions that provide such a site. Viomycin binds to the full-length IRES and RNA fragments with K(d) values of 25-55 microM. Interestingly, viomycin binding to the two fragments is affected differently by Mg(2+); noncompetitive inhibition of binding to 3DEF4 is observed, whereas binding to 3EF4 is not impaired. Formation of a Mg(2+)-stabilized tertiary fold, involving subdomain IIId, may thereby hinder viomycin binding to 3DEF4 indirectly. Mutational and deletion studies locate viomycin binding within subdomains IIIe/f rather than within the pseudoknot. In pseudoknot mutants, Mg(2+) ions have different effects on viomycin binding and thermal stability, suggesting altered tertiary interactions involving subdomain IIId.