Binding of yeast tRNAPhe anticodon arm to Escherichia coli 30 S ribosomes

A 15-nucleotide fragment of RNA having the sequence of the anticodon arm of yeast tRNAPhe was constructed using T4 RNA ligase. The stoichiometry and binding constant of this oligomer to poly(U)-programmed 30 S ribosomes was found to be identical to that of deacylated tRNAPhe. The anticodon arm and tRNAPhe also compete for the same binding site on the ribosome. These data indicate that the interaction of tRNAPhe with poly(U)-programmed 30 S ribosomes is primarily a result of contacts in the anticodon arm region and not with other parts of the transfer RNA. Since similar oligomers which cannot form a stable helical stem do not bind ribosomes, a clear requirement for the entire anticodon arm structure is demonstrated.     

NMR studies of ion binding to Escherichia coli tRNAPhe

The effects of magnesium, spermine, and temperature on the conformation of Escherichia coli tRNAPhe have been examined by proton and phosphorus nuclear magnetic resonance spectroscopy. In the low-field proton NMR spectra we have characterized two slowly interconverting conformations of this tRNA at low magnesium ion concentrations. The relative proportion of the conformers is ion dependent but not ion specific. Magnesium affects protons in all the stems of tRNA while spermine effects are localized near the s4U-8-A-14 and G-15-C-48 tertiary bonds. The effects seen in the proton NMR spectra are compared and correlated with those observed in the phosphorus spectra to give assignments of some of the resolved signals from the phosphate groups. The phosphorus spectra are compared with those of yeast tRNAPhe [Gorenstein, D. G., Goldfield, E. M., Chen, R., Kovar, K., & Luxon, B. A. (1981) Biochemistry 20, 2141; Salemink, P. J. M., Reijerse, E. J., Mollevanger, L., & Hilbers, C. W. (1981) Eur. J. Biochem. 115, 635], and the ion effects are discussed with reference to the magnesium and spermine sites found in the crystal structures of yeast tRNAPhe [Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, S.-H. (1977) Nucleic Acids Res. 4, 2811; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64; Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315].     

Interactions of yeast tRNAPhe with ribosomes from yeast and Escherichia coli. A fluorescence spectroscopic study.

The interaction of ethidium-labeled tRNAPhe from yeast with ribosomes from yeast and Escherichia coli was studied by stead-state measurements of fluorescence intensity and polarization. The ethidium label was covalently inserted into either the anticodon or the dihydrouridine loop of the tRNA. The codon-independent formation of a tRNA-ribosome complex led to only a moderate increase of the observed fluorescence polarization indicating a considerable internal mobility of the labeled parts of the tRNA molecule in the ribosome complex. When the ribosome complex was formed in the presence of poly(U), the probes both in the dihydrouridine loop and in the anticodon loop were strongly immobilized, the latter exhibiting a substantial increase in fluorescence intensity. A smaller intensity change was observed when E. coli ribosomes were used, although the extent of immobilization was found to be similar in this case. Competition experiments with non-labeled tRNAPhe showed that the labeled tRNAPheEtd was readily released from the complex with yeast ribosomes when poly(U) was absent, whereas in the presence of poly(U) it was bound practically irreversibly. The finding that the mobility of a probe in the dihydrouridine loop is affected by the codon-anticodon interaction on the ribosome suggests a conformational change of the ribosome-bound tRNA which may involve opening of the tertiary structure interactions between the dihydrouridine and the TpsiC loop.     

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.     

Interaction of a fluorescent streptomycin derivative with Escherichia coli ribosomes.

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

Role of the constant uridine in binding of yeast tRNAPhe anticodon arm to 30S ribosomes.

Twenty-two anticodon arm analogues were prepared by joining different tetra, penta, and hexaribonucleotides to a nine nucleotide fragment of yeast tRNAPhe with T4 RNA ligase. The oligomer with the same sequence as the anticodon arm of tRNAPhe bind poly U programmed 30S ribosomes with affinity similar to intact tRNAPhe. Analogues with an additional nucleotide in the loop bind ribosomes with a weaker affinity whereas analogues with one less nucleotide in the loop do not bind ribosomes at all. Reasonably tight binding of anticodon arms with different nucleotides on the 5' side of the anticodon suggest that positions 32 and 33 in the tRNAPhe sequence are not essential for ribosome binding. However, differences in the binding constants for anticodon arms containing modified uridine residues in the "constant uridine" position suggest that both of the internal "U turn" hydrogen bonds predicted by the X-ray crystal structure are necessary for maximal ribosome binding.     

Characterisation of the binding of virginiamycin S to Escherichia coli ribosomes.

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50-S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting-reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50-S subunit.     

Neutron scattering study of the binding of tRNAPhe to Escherichia coli phenylalanyl-tRNA synthetase

Escherichia coli phenylalanyl-tRNA synthetase has been characterized by small-angle neutron scattering. In solution (20 mM imidazole hydrochloride, pH 7.6, 10 mM 2-mercaptoethanol, and 0.1 mM ethylenediaminetetraacetic acid), this enzyme has a molecular weight of 227K +/- 20K with a radius of gyration of 48.3 +/- 0.6 A, independent of the presence of MgCl2 up to 50 mM. The change of the scattering upon adding tRNAPhe to the enzyme has been followed with 10 mM MgCl2 present in the buffer. One enzyme molecule is capable of binding two tRNAPhe molecules with affinity constants larger than 10(6) M-1. Parallel titration experiments in 73% 2H2O, close to the matching point of tRNA, show that the RG of the enzyme is not changed by the binding of one or two tRNAPhe molecules. These results are compared with quasi-electric light scattering studies [Holler, E., Wang, C. C., & Ford, N.C., Jr. (1981) Biochemistry 20, 861-867] where the addition of either MgCl2 or tRNAPhe was shown to cause dramatic changes of the apparent translational diffusion constant of phenylalanyl-tRNA synthetase.     

Investigating the entire course of telithromycin binding to Escherichia coli ribosomes

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (K(T) = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl-aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (K(T)* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.     

On the structural specificity of puromycin binding to Escherichia coli ribosomes

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.     

Magnesium binding to yeast ribosomes

This paper describes a theoretical and experimental analysis of the binding of magnesium ions to yeast, ribosomes. In the theoretical considerations the interactions between charges located on a macroion are included. In the calculations these interactions result in a term, in which both the charge and the radius of the macroion are accounted for. It appears that on dissociation of the ribosomes both the charge and the radius change, but in such a way, that the term, which accounts for the electrostatic interactions, remains constant. As a consequence the dissociation can lie neglected in the analyses of the binding experiments. Our experiments indicate that two binding reactions between ribosomes and magnesium ions occur. The endpoints of these reactions correspond to about 0.40 and 1.0 equivalent magnesium per ribosomal phosphate, respectively. The pK values are about 3.8 and 2.2, respectively. The experimental results indicate that the effect, of monovalent cations can be explained as a pure ionic strength effect, though the binding of monovalent cations could not be excluded completely.     

tRNA binding sites of ribosomes from Escherichia coli

70S tight-couple ribosomes from Escherichia coli were studied with respect to activity and number of tRNA binding sites. The nitrocellulose filtration and puromycin assays were used both in a direct manner and in the form of a competition binding assay, the latter allowing an unambiguous determination of the fraction of ribosomes being active in tRNA binding. It was found that, in the presence of poly(U), the active ribosomes bound two molecules of N-AcPhe-tRNAPhe, one in the P and the other in the A site, at Mg2+ concentrations between 6 and 20 mM. A third binding site in addition to P and A sites was observed for deacylated tRNAPhe. At Mg2+ concentrations of 10 mM and below, the occupancy of the additional site was very low. Dissociation of tRNA from this site was found to be rather fast, as compared to both P and A sites. These results suggest that the additional site during translocation functions as an exit site, to which deacylated tRNA is transiently bound before leaving the ribosome. Since tRNA binding to this site did not require the presence of poly(U), a function of exit site bound tRNA in the fixation of the mRNA appears unlikely. Both the affinity and stability of binding to the additional site were found lower for the heterologous tRNAPhe from yeast as compared to the homologous one. This difference possibly indicates some specificity of the E. coli ribosome for tRNAs from the same organism.     

Protein involved in the binding of dihydrostreptomycin to ribosomes of Escherichia coli

At increasing ammonium chloride concentrations, 30 S subunits on one hand, and 50 S subunits, 16 S BNA and 23 S RNA on the other hand, show a different behaviour with respect to dihydrostreptomycin binding. Within a wide range (10 to 250 mm) binding to 30 S subunits is not affected by NH₄Cl, whereas binding to 50 S and the RNAs decreases by increasing NH₄Cl concentrations. 30 S subunits lose more than 90% of their binding capacity by washing with 1.15 m-LiCl (SP_1.5³).The split proteins SP_1.15 were analysed by DEAE-cellulose chromatography and Sephadex G100 gel filtration. After reconstitution with the non-binding 2.0 core the proteins S3 and S5 can bind dihydrostreptomycin independently of each other; the S5-dependent binding is stimulated by S9 and S14 (S10). The Scatchard plot revealed 0.8 binding sites per 30 S subunit. We conclude that S3 and S5 are part of one binding site of dihydrostreptomycin.     

Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

In previous work we showed that on photolysis of Escherichia coli ribosomes in the presence of [3H]tetracycline (TC) the major protein labeled is S7, and we presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC [Goldman, R. A., Hasan, T., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1983) Biochemistry 22, 359-368]. In this work we use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays. With respect to both sedimentation coefficients and relative Phe-tRNAPhe binding, the properties of the SPORE particles we obtain parallel very closely those measured earlier [Nomura, M., Mizushima, S., Ozaki, M., Traub, P., & Lowry, C. V. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 49-61], with the exception of the SPORE particle lacking S13. A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14. Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another. A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1396, in one of the major domains of the 30S subunit.