Paromomycin and dihydrostreptomycin binding to Escherichia coli ribosomes.

Paromomycin binds specifically to a single type of binding site on the 70-S streptomycin-sensitive Escherichia coli ribosome. This site is different from that of dihydrostreptomycin since paromomycin binds to streptomycin-resistant ribosomes and sine dihydrostreptomycin does not compete for paromomycin binding. Paromomycin binding, unlike dihydrostreptomycin binding, is independent of changes in ribosome concentration but influenced by magnesium ion concentration. Moreover, paromomycin does not bind to the 30-S subunit of the streptomycin-sensitive ribosome, except in the presence of dihydrostreptomycin, which probably induces the conformational changes necessary for a paromomycin binding site. This induction does not occur with streptomycin-resistant ribosomes. Neither antibiotic binds to the 50-S subunit. In general, binding of the one antibiotic increases the number of sites available for binding of the other. Both antibiotics exhibit marked non-specific binding at high antibiotic/ribosome ratios. Competition studies have enabled the classification of other aminoglycosides according to their ability to compete for the paromomycin and dihydrostreptomycin binding sites. Derivatives structurally related to paromomycin compete for its binding, the degree of competition being related to antibacterial activity, but do not compete for dihydrostreptomycin binding; they, on the contrary, increase the number of dihydrostreptomycin binding sites. Neither gentamicin nor kanamycin derivatives, which induce a high level of misreading, nor kasugamycin and spectinomycin, which do not induce misreading, compete for paromomycin or dihydrostreptomycin binding sites. Other sites may be involved in the binding of these aminoglycosides and in inducing misreading.     

Mechanism of action of aminoglycoside antibiotics. Binding studies of tobramycin and its 6'-N-acetyl derivative to the bacterial ribosome and its subunits.

6'-N-[14C]Acetyl-tobramycin and [3H]tobramycin were synthesized and their binding to Escherichia coli ribosomes and ribosomal subunits studied using equilibrium dialysis. THE 70-S ribosome, as well as its 50-S and 30-S subunits, bound tightly to 6'-N-acetyl-tobramycin. The binding of [3H]tobramycin to ribosomes was quite different. The 70-S ribosome was observed to possess several classes of binding sites; of these, one was determined to be of higher affinity and lower capacity, the 6'-N-[14C]acetyl-tobramycin site. The isotopic dilution method was used to define the specificity of the interaction. The selective binding of 6'-N-[14C]acetyl-tobramycin was highly reversible by tobramycin, kanamycins A, B, C and neomycin, but not by streptomycin or erythromycin. Gentamicin C1a was a poor inhibitor. This suggested that either the kanosamin or garosamin rings might be determinant in the binding of these molecules, as well as the 6'-amino group.     

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.     

The effect of initiation factor IF-1 on the dissociation of 70-S ribosomes of Escherichia coli.

By means of exchange studies, in which 3H-labelled 50-S subunits and unlabelled 70-S ribosomes from Escherichia coli MRE 600 were used, it has been demonstrated that the 30-S subunit is the only target for IF-3 in the dissociation of 70-S ribosomes. The interference of IF-3 with the dynamic equilibrium of 70-S in equilibrium 50-S + 30-S occurs by binding of the factor to the 30-S subunit. The 30-S-IF-3 complex in impaired in the association reaction, which implies that IF-3 is acting as an anti-association factor. The action of IF-1 is two-fold. Firstly IF-1 increases the rate of exhcange of the ribosomal subparticles in the 70-S ribosome without changing the position of the equilibrium. Thus the spontaneous equilibrium is attained more rapidly in the presence of IF-1. This kinetic effect of IF-1 is also demonstrated in the IF-3-mediated dissociation of 70-S ribosomes. Secondly IF-1 is able to increase the IF-3-mediated dissociation. It seems likely that the explanation for the latter phenomenon must be sought in the binding of IF-1 to 70-S ribosomes, resulting in a loosening of the ribosomes structure, as well as to 30-S. IF-3 complex, thaereby slowing down the association reactions of the subunits.     

Functional interaction of neomycin B and related antibiotics with 30S and 50S ribosomal subunits.

Antibiotics of the neomycin, kanamycin and gentamicin, but not streptomycin, groups stabilize the GDP·elongation factor (EF) G·50S subunit·fusidic acid complex. Treatment of 30S subunits, but not of 50S subunits, with neomycin B or kanamycin B, followed by removal of excess unbound antibiotic and supplementation with untreated complementary subunits, promotes poly(U)-dependent binding of Tyr-tRNA to the reassociated ribosomes (misreading). A similar treatment of either ribosomal subunit with neomycin B inhibits the EF-G-dependent translocation of Ac-Phe-tRNA. These results suggest that interaction of neomycin B and related antibiotics with the 30S subunit induces misreading and inhibits translocation, and interaction with the 50S subunit stabilizes EF-G on the ribosome and also inhibits translocation.     

Initiation factor IF-3 and the binary complex between initiation factor IF-2 and formylmethionyl-tRNA are mutually exclusive on the 30-S ribosomal subunit.

The formation of 30-S initiation complexes depends strongly on initiation factor IF-3; at molar ratios of IF-3 to 30-S ribosomes up to one a stimulation is observed, whereas at ratios higher than one, initiation complex formation declines strongly. The target of the observed inhibition of fMet-tRNA binding at high concentrations of IF-3 is the 30-S initiation complex itself. On the one hand addition of IF-3 to preformed 30-S initiation complexes leads to a release of bound fMet-tRNA which is linear with the amount of factor added, whereas no effect on isolated 70-S initiation complexes is seen. The release of fMet-tRNA from preformed 30-S initiation complexes is accompanied by a release of IF-2 in a one-to-one molar ratio which is in agreement with our previous findings showing that binding of fMet-tRNA takes place via a binary complex: IF-2 . fMet-tRNA (Eur. J. Biochem. 66, 181--192 and 77, 69--75). On the other hand increasing amounts of both IF-2 and fMet-tRNA relieve the IF-3-induced inhibition of 30-S initiation complex formation. From these findings it is concluded that IF-3 and the IF-2 . fMet-tRNA complex are mutually exclusive on the 30-S ribosome. This implies that under our experimental conditions MS2 RNA binding precedes fMet-tRNA binding if one accepts that the presence of IF-3 on the 30-S subunit is obligatory for messenger binding.     

Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation.

The binding of [${}^3\text{H}$]kanamycin to $\text{E. coli}$ ribosomes and ribosomal subunits was studied by equilibrium dialysis and Millipore filter methods. The 70S ribosome bound ca. two molecules up to the antibiotic concentration of 10 uM, and more at higher concentrations. Each ribosomal subunit was observed to possess one major binding site, and the affinity of the small ribosomal subunit was greater than that of the large subunit. The binding of [${}^3\text{H}$]kanamycin to ribosomes and ribosomal subunits was reversed by neomycin or gentamicin, but not by streptomycin and chloramphenicol. Kanamycin, neomycin and gentamicin interfered with the binding of [${}^{14}\text{C}$] tuberactinomycin O. Translocation of N-Ac-Phe-tRNA was markedly inhibited by kanamycin, neomycin or gentamicin, but not by streptomycin.     

Binding of tRNA in different functional states to Escherichia coli ribosomes as measured by velocity sedimentation

The binding of initiator and elongator tRNAs to 70-S ribosomes and the 30-S subunits was followed by velocity sedimentation in the analytical ultracentrifuge. fMet-tRNAfMet binds to A-U-G-programmed 30-S subunits, but not to free or misprogrammed particles. Both the formylmethione residue and the initiation factors increase the stability of the 30-S x A-U-G x fMet-tRNAfMet complex. fMet-tRNAfMet is bound only to the P site of the 70-S ribosome even in the absence of A-U-G. Two copies of tRNAPhe or Phe-tRNAPhe are bound to the ribosome with similar affinity. In contrast to a recent report [Rheinberger et al. (1981) Proc. Natl Acad. Sci. USA, 78, 5310-5314], it is shown that three copies of tRNA cannot be bound simultaneously to the ribosome with binding constants higher than 2 x 10(4) M-1. Phe-tRNAPhe when present as the ternary complex Phe-tRNAPhe. EF-Tu x guanosine 5'-[beta,gamma-methylene]triphosphate binds exclusively to the A site. The peptidyl-tRNA analogue, acetylphenylalanine-tRNA, can occupy both ribosomal centers, albeit with a more than tenfold higher affinity for the P site. The thermodynamic data obtained under equilibrium conditions confirm the present view of two tRNA binding sites on the ribosome. The association constants determined are discussed in relation to the mechanism of ribosomal protein synthesis.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes. Role of 50 S subunits in formation of the E site

The 30 S subunit contains 2 sites for tRNA binding (Phe-tRNA, AcPhe-tRNA, tRNAPheOH) with the functional properties of D and A sites of the 70 S ribosome after attachment of 50 S subunit. The third (E) site specific for deacylated tRNA is introduced into 70 S ribosome by its 50 S subunit. The E-site binding of tRNAPheOH is not sensitive to either tetracycline and edeine, and practically codon-independent. The affinity constant of tRNAPheOH for the E site is 2-3 orders of magnitude lower than that for the D site.     

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.     

Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.     

Identification of the amino acid functional groups responsible for 30-S ribosome recognition of messenger RNA.

About 30 protein-selective chemical reagents have been tested for their ability to inhibit the mRNA binding activity of the 30-S ribosome. A number of reagents were investigated which have been shown by other workers to be capable of modifying free epsilon-amino groups of lysine and all were found to inactivate 30-S ribosomes completely for natural mRNA binding activity. Several reagents selective for histidine, tyrosine, and tryptophan were also found to inactivate. We suggest that the epsilon-amino groups of lysine play an important role in mRNA binding to the 30-S ribosome.     

Structural basis for hygromycin B inhibition of protein biosynthesis

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.     

Effect of viomycin on dihydrostreptomycin binding to bacterial ribosomes.

Viomycin, a peptide antibiotic, reduced the amounts of dihydrostreptomycin bound to ribosomes of Myobacterium smegmatis and Escherichia coli, although they have different modes of action. The [3H]dihydrostreptomycin binding to ribosomes could not exchanged with streptomycin or dihydrostreptomycin, but not with unrelated antibiotics, namely, kanamycin, neomycin, spectinomycin, capreomycin, tuberactinomycin-N, chloramphenicol and erythromycin. We suggest that there is a significant interaction between the binding sites of viomycin and streptomycin on ribosomes.     

The context theory as applied to the decoding of the initiator tRNA by Escherichia coli ribosomes

The involvement of nucleotides adjacent to the termination codons in tRNA during the suppression of termination has been formulated as the 'context theory' by Bossi and Roth (1980) [Nature (Lond.) 286, 123-127]. The finding that U-U-G functions as an initiator codon has revived the discussion on the participation of the nucleotides flanking the initiator triplet in the decoding of initiator tRNA (context theory of initiation by the ribosome). We compared the capacity of oligonucleotides cognate to the anticodon loop of formylmethionine tRNA, such as A-U-G, A-U-G-A and U-A-U-G-A, to enhance the formation of the 30-S and 70-S ribosomal initiation complexes. Three different methods were used to determine the apparent binding constants and the stoichiometries of the respective complexes: adsorption of the complexes to nitrocellulose filters, equilibrium dialysis, and velocity sedimentation. We found that in the 30-S ribosomal initiation complex and in the presence of initiation factor 2 and GTP, formylmethionyl-tRNA is preferentially decoded by more than three mRNA bases. With the 70-S ribosome, however, once initiation factor 2 had been released, A-U-G represented the most effective codon to direct the formylmethionyl-tRNA to the peptidyl site. An extended initiator sequence may either give additional stability to the 30-S initiation complex or may allow for an ambiguity by one base pair in the decoding of the initiator tRNA.     

Chemical inactivation of Escherichia coli 30-S ribosomes by iodination. Identification of proteins involved in tRNA binding.

30-S ribosomal subunits are inactivated by iodination for both enzymic fMet-tRNA and non-enzymic Phe-tRNA binding activities. This inactivation is due to modification of the protein moiety of the ribosome. Reconstitutions were performed with 16-S RNA and mixtures of total protein isolated from modified subunits and purified proteins isolated from unmodified subunits. This allowed identification of the individual proteins which restore tRNA binding activity. S3, S14 and S19 were identified as proteins involved in fMet-tRNA binding. S1, S2, S3, S14 and S19 were identified as proteins involved in Phe-tRNA binding. Modified particles shown normal sedimentation constants and complete protein compositions both before and after reconstitution. This suggests that the loss of activity is due to modification of one or more of the actual binding sites located on the 30-S subunit and that restoration of activity is due to structural correction at this site rather than to correction of an assembly defect.     

Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy

Poly(U) with an average chain length of 40-70 nucleotides was modified at the 5'- or 3'-terminal residues with 2,4-dinitrophenyl derivatives. The modified poly(U) was used to form 30S.poly(U) or 70S.poly(U).Phe-tRNA complexes. Localization of the 5' and 3' ends of the template polynucleotide on the 30S subunit and the 70S ribosome was performed by immune electron microscopy using antibodies against dinitrophenyl haptens. The 5' and 3' ends of poly(U) (putative entry and exit sites of the message) were found in the same region both on the 30S subunit and the 70S ribosome. They were located on the dorsal side of the 30S subunit between the head and the body near the groove bordering the side ledge (platform). Comparison of the size of this region with the possible length of the polynucleotide chain covered by the ribosome allowed us to suggest that the message makes a 'U-turn" (or forms a 'loop') as it passes through the ribosome.     

Localization of the protein L2 in the 50 S subunit and the 70 S E. coli ribosome

The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex. The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation. L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit. The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix. The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs.     

Ribosomal proteins directly interacting with fMet-tRNAfMet in the 30S initiation complex

By means of ultraviolet-induced (254nm) RNA-protein cross-links it is shown, that tRNAfMet inside the preinitiation complex, formed by binding of fMet-tRNAfMet with 30S subunit of E. coli ribosome and RNA of the phage MS2 in the presence of initiation factors, directly interacts with proteins S4, S5, S9, S11, S14 and S15-S17.