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.     

Composition of the Escherichia coli 70S ribosomal interface: a cross-linking study

70S tight-couple ribosomes from Escherichia coli were cross-linked by using the bifunctional reagent phenyl-diglyoxal (PDG). The reaction was stopped after 4-h incubation while still in the linear range. In comparison with untreated ribosomes, 30% of those treated with PDG were shown, by sucrose gradient experiments, not to be separable into their subunits, but remained as 70S particles. There was no detectable change in the structure of the reacted particles when their sedimentation behavior was compared with that of native 70S controls. When the cross-linking reaction was performed in the presence of tRNAPhe and poly(U), the reacted ribosomes retained 40-50% of their tRNA binding activity. The reaction leads predominantly to the formation of RNA-protein cross-links but protein--protein as well as RNA-RNA cross-links could also be detected. Cross-linked material was extracted, and the individual RNAs were separated into 23S, 16S, and 5S RNAs. Proteins were identified electrophoretically after reversal of the RNA-protein cross-links. Proteins were found to be cross-linked to RNAs within and across the ribosomal subunits; the latter are considered to be close to or at the 70S subunit interface. The arrangement of RNA and protein at the subunit interface is discussed.     

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.     

The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.     

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.     

Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes

Binding of tRNAPhe to ribosomes shields a set of highly conserved nucleotides in 16S rRNA from attack by a combination of structure-specific chemical probes. The bases can be classified according to whether or not their protection is strictly poly(U)-dependent (G529, G530, U531, A1408, A1492, and A1493) or poly(U)-independent (A532, G693, A794, C795, G926, 2mG966, G1338, A1339, U1381, C1399, C1400, and G1401). A third class (A790, G791, and A909) is shielded by both tRNA and 50S ribosomal subunits. Similar results are obtained when the protecting ligand is tRNAPhe E. Coli, tRNAPhe yeast, tRNAPhe E. Coli lacking its 3' terminal CA, or the 15 nucleotide anticodon stem-loop fragment of tRNAPhe yeast. Implications for structural correlates of the classic ribosomal A- and P-sites and for the possible involvement of 16S rRNA in translational proofreading are discussed.     

40 S subunits from rat liver ribosomes contain two codon-dependent sites for transfer RNA

40 S subunits from rat liver ribosomes are able to bind, after heat activation, two molecules of either Phe-tRNAPhe, Ac-Phe-tRNAPhe or deacylated tRNAPhe. Addition of 60 S subunits to the quaternary complex 40 S.poly(U).(Phe-tRNAPhe)2 results in quantitative formation of (Phe)2-tRNAPhe. This indicates that the two binding sites for tRNA on 40 S subunits should be considered as the constituent of P and A sites of 80 S ribosomes.     

Proton nuclear magnetic resonance study of the effect of pH on tRNA structure.

The low-field 220-MHz proton nuclear magnetic resonance (NMR) spectra of four tRNA molecules, Escherichia coli tRNAPhe, tRNA1Val, and tRNAfMet1, and yeast tRNAPhe, at neutral and mildly acidic pH are compared. We find a net increase in the number of resonances contributing to the -9.9-ppm peak (downfield from sodium 4,4-dimethyl-4-silapentanesulfonate) in three of these tRNAs at pH 6, while tRNAfMet1 does not clearly exhibit this behavior. The increase in intensity at this resonance position is half-completed at pH 6.2 in the case of yeast tRNAPhe. An alteration at the 5'-phosphate terminus is not involved, since removal of the terminal phosphate does not affect the gain in intensity at -9.9 ppm. Based on a survey of the tertiary interactions in the four molecules, assuming that they possess tertiary structures like that of yeast tRNAPhe at neutral pH, we tentatively attribute this altered resonance in E. coli and yeast tRNAPhe to the protonation of the N3 of the adenine residue at position 9 which results in the stabilization of the tertiary triple A23-U12-A9. This intepretation is supported by model studies on the lowfield proton NMR spectrum of AN oligomers at acid pH, which reveal an exchanging proton resonance at -9.4 ppm if the chain length N greater than or equal to 6.     

The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.     

tRNA binding sites on the subunits of Escherichia coli ribosomes

Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity. Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA. Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent. The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies. Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site). Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes. In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA. Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site. The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes. 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits. The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.     

Effect of modeccin on the steps of peptide-chain elongation.

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.     

Poly(U)-dependent interaction of yeast tRNA(Phe) and its fragments with ribosomes from Escherichia coli

The poly(U)-dependent bindings of yeast tRNAPhe, its derivative depleted of 3'-terminal adenosine, and 15-nucleotide having a sequence of yeast tRNAPhe anticodon arm to the P site of Escherichia coli 70S ribosomes were compared. The equilibrium and rate constants were determined. Data indicate that the anticodon arm (N28-N42) contributes the major fraction of the binding free energy (-45.3 kJ/mol at 10 mM Mg2+ and 30 degrees C). Other parts of the tRNAPhe molecule besides A76 (N1-N27 and N43-N75) bring additional-6.0 kJ/mol, and A76 contributes-2.4 kJ/mol.     

Effect of ribosome binding and translocation on the anticodon of tRNAPhe as studied by wybutine fluorescence.

The complexes of N-AcPhe-tRNAPhe (or non-aminoacylated tRNAPhe) from yeast with 70S ribosomes from E. coli have been studied fluorimetrically utilizing wybutine, the fluorophore naturally occurring next to the 3' side of the anticodon, as a probe for conformational changes of the anticodon loop. The fluorescence parameters are very similar for tRNA bound to both ribosomal sites, thus excluding an appreciable conformational change of the anticodon loop upon translocation. The spectral change observed upon binding of tRNAPhe to the P site even in the absence of poly(U) is similar to the one brought about by binding of poly(U) alone to the tRNA. This effect may be due to a hydrophobic binding site of the anticodon loop or to a conformational change of the loop induced by binding interactions of various tRNA sites including the anticodon.     

15N-labeled tRNA. Identification of dihydrouridine in Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR

The N3 imino units of dihydrouridine were identified in samples of 15N-labeled Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. The peaks for dihydrouridine had high field 1H (9.7-9.8 ppm) and 15N (147.8-149.5 ppm) chemical shifts. Assignments were made by 1H-15N chemical shift correlation based on values obtained in model studies with tri-O-benzoyl- and tri-O-acetyldihydrouridine. The rates of exchange of the imino protons with water suggest that the D-loop in tRNAfMet is less stable than the D-loops in tRNALys or tRNAPhe. Closely spaced peaks were observed for the two dihydrouridines in tRNAPhe in a high resolution spectrum.     

Recognition of tRNA Trp by initiation factors from Escherichia coli.

Binding of acetyl or formyltryptophanyl-tRNA Trp from Escherichia coli or beef liver to E. coli ribosomes is strongly stimulated by E. coli initiation factors and requires GTP. The N-acylated tryptophan is puromycin reactive. Polypeptide chain initiation with acetyltryptophan dependent on poly(U,G) has been demonstrated and is highly dependent on added initiation factors. tRNA Trp appears, therefore, to share some structural features with tRNAfMet of significance to the process of polypeptide chain initiation.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Number of tRNA binding sites on 80 S ribosomes and their subunits

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5-20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA.     

Replacement of pseudouridine in transfer RNA by 5-fluorouridine does not affect the ability to stimulate the synthesis of guanosine 5'-triphosphate 3'-diphosphate.

The requirement for ribothymidine and pseudouridine in the TpsiCG loop of tRNA for its activity in the ribosome and tRNA-stimulated synthesis of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) by stringent factor has been tested by the use of a purified tRNAPhe (883 pmol of phenylalanine incorporated/A260 unit) in which 92% of the pseudouridine, 98% of the ribothymidine, 98% of the dihydrouridine, and 88% of the uridines were substituted by 5-fluorouridine. This tRNA was quantitatively as active as control tRNA in inducing pppGpp synthesis. With loose-couple ribosomes, the concentration of tRNA needed to give half-maximal reaction was 0.07 micrometer for both normal and fluorouridine-substituted tRNA, with vacant tight-couple ribosomes it was 0.05 micrometer, and with tight couples carrying poly(Phe)-tRNA at the P site the value was 0.02 micrometer. These results show that at the level of intact tRNA there is no special requirement for modified bases in the TpsiCG loop of tRNA in the synthesis of pppGpp.     

Biochemical characterization and messenger specificity of polypeptide chain initiation factors from Escherichia coli

Three protein factors are required for maximum poly(U, G)- or AUG-directed binding of fMet-tRNA to ribosomes. The same three factors are both necessary and sufficient for “natural” mRNA-directed binding of fMet-tRNA to ribosomes. Bound fMet-tRNA cosediments with the 70S ribosome as does bound mRNA. All three factors are required for the fMet-tRNA and GTP-dependent binding of mRNA to the 70S initiation complex.     

Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome

A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes. The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques. The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment. The cDNA probe was found to interact specifically with its rRNA target site. The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated. The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes. Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes. These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction.