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.     

Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two codon-specific binding sites for transfer RNA.

30S subunits were isolated capable to bind simultaneously two molecules of Phe-tRNAPhe (or N-Acetyl-Phe-tRNAPhe), both poly(U) dependent. The site with higher affinity to tRNA was identified as P site. tRNA binding to this site was not inhibited by low concentrations of tetracycline (2 x 10(-5)M) and, on the other hand, N-Acetyl-Phe-tRNAPhe, initially prebound to the 30S.poly(U) complex in the presence of tetracycline, reacted with puromycin quantitatively after addition of 50S subunits. The site with lower affinity to tRNA revealed features of the A site: tetracycline fully inhibited the binding of both Phe-tRNAPhe and N-Acetyl-Phe-tRNAPhe. Binding of two molecules of Phe-tRNAPhe to the 30S.poly(U) complex followed by the addition of 50S subunits resulted in the formation of (Phe)2-tRNAPhe in 75-90% of the reassociated 70S ribosomes. These results prove that isolated 30S subunits contain two physically distinct centers for the binding of specific aminoacyl- (or peptidyl-) tRNA. Addition of 50S subunits results in the formation of whole 70S ribosomes with usual donor and acceptor sites.     

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.     

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.     

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.     

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.     

Ribosome binding by tRNAs with fluorescent labeled 3'' termini.

Yeast and E. coli tRNAPhe samples were oxidized and labeled at the 3' end with dansyl hydrazine or fluorescein thiosemicarbazide. These tRNAs can bind to poly(U)-programmed E. coli 70S tight couple ribosomes in 25 mM magnesium at 8 degrees C. Two binding sites with binding constants of about 1 X 10(9) M-1 (P) and 3 X 10(7) M-1 (A) were determined for the yeast tRNAPhe derivatives. With E. coli tRNAPhe the A site affinity is similar to yeast tRNAPhe but the P site affinity is 5-fold weaker. Singlet-singlet energy transfer showd that the distance from the 3' end of tRNAPhe in the P site to a fluorescein derivative of erythromycin is 23 A. This supports in vitro studies suggesting that erythromycin binds near the peptide moiety of peptidyl tRNA. A distance of 34 A between the 3' ends of 2 tRNAs bound simulatneously on the ribosome was also measured. This long distance may mean that the deacylated fluorescent tRNA binds to the A site in an orientation like that in the stringent response rather than in protein synthesis.     

Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center

Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.     

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.     

Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers.

Interactions between Escherichia coli 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to cDNA oligomers, regions 517-528, 1397-1404, and 1534-1542. The binding of cDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNAPhe. Only the binding of cDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAPhe or tRNAfMet alone. The poly(AGU)/tRNAfMet ligand pair caused an additional decline in the binding of cDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the cDNA oligomers 517-528 or 1398-1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for cDNA 1534-1541 in the presence of mRNAs or tRNA. At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other ligands, caused a large decrease in the binding of the cDNA oligomers. The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight-couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [1].     

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.     

Codon-anticodon interaction at the ribosomal E site

The question of whether or not the tRNA at the third ribosomal binding site specific for deacylated tRNA (E site) undergoes codon-anticodon interaction was analyzed as follows. Poly(U)-programmed ribosomes each carrying two [14C]tRNAPhe molecules were subjected to a chasing experiment using various tRNA species. At 0 degree C Ac[3H]Phe-tRNAPhe did not trigger any chasing whereas deacylated cognate tRNAPhe provoked a strong effect; non-cognate tRNALys was totally ineffective. This indicates that the second [14C]tRNAPhe cannot be present at the A site but rather at the E site (confirming previous observations). In the presence of poly(U) or poly(A) ribosomes bound the cognate tRNA practically exclusively as second deacylated tRNA, i.e. [14C]tRNAPhe and [14C]tRNALys, respectively. Thus, the second deacylated tRNA binds in a codon-dependent manner. [14C]tRNALys at the P site and Ac[3H]Lys-tRNALys at the A site of poly(A)-primed ribosomes were translocated to the E and P sites, respectively, by means of elongation factor G. The E site-bound [14C]tRNALys could be significantly chased by cognate tRNALys but not by non-cognate tRNAPhe, indicating the coded nature of the E site binding. Additional evidence is presented that the ribosome accommodates two adjacent codon-anticodon interactions at either A and P or P and E sites.     

Mechanism of ribosomal translocation. Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis

The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions. A burst of GTP hydrolysis, with a kapp value greater than 30 s-1 (20 degrees C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid. The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid. E. coli tRNAPhe, but not N-acetylphenylalanyl-tRNAPhe (N-AcPhe-tRNAPhe), stimulates the GTPase when bound in the P site. If the A site of poly(U)-programmed ribosomes, carrying tRNAPhe in the P site, is occupied by N-AcPhe-tRNAPhe, the burst of Pi discharge is replaced by a slow GTP hydrolysis. Since, under these conditions, N-AcPhe-tRNAPhe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.     

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.     

Affinities of tRNA binding sites of ribosomes from Escherichia coli

The binding affinities of tRNAPhe, Phe-tRNAPhe, and N-AcPhe-tRNAPhe from either Escherichia coli or yeast to the P, A, and E sites of E. coli 70S ribosomes were determined at various ionic conditions. For the titrations, both equilibrium (fluorescence) and nonequilibrium (filtration) techniques were used. Site-specific rather than stoichiometric binding constants were determined by taking advantage of the varying affinities, stabilities, and specificities of the three binding sites. The P site of poly(U)-programmed ribosomes binds tRNAPhe and N-AcPhe-tRNAPhe with binding constants in the range of 10(8) M-1 and 5 X 10(9) M-1, respectively. Binding to the A site is 10-200 times weaker, depending on the Mg2+ concentration. Phe-tRNAPhe binds to the A site with a similar affinity. Coupling A site binding of Phe-tRNAPhe to GTP hydrolysis, by the addition of elongation factor Tu and GTP, leads to an apparent increase of the equilibrium constant by at least a factor of 10(4). Upon omission of poly(U), the affinity of the P site is lowered by 2-4 orders of magnitude, depending on the ionic conditions, while A site binding is not detectable anymore. The affinity of the E site, which specifically binds deacylated tRNAPhe, is comparable to that of the A site. In contrast to P and A sites, binding to the E site is labile and insensitive to changes of the ionic strength. Omission of the mRNA lowers the affinity at most by a factor of 4, suggesting that there is no efficient codon-anticodon interaction in the E site. On the basis of the equilibrium constants, the displacement step of translocation, to be exergonic, requires that the tRNA leaving the P site is bound to the E site. Under in vivo conditions, the functional role of transient binding of the leaving tRNA to the E site, or a related site, most likely is to enhance the rate of translocation.     

Structure of a novel flavin chromophore from Avena coleoptiles, the possible ''blue light'' photoreceptor

The effect of edeine on the translation of mRNA or poly(U)-directed polyphenylalanine synthesis has been studied in an edeine-resistant mutant of Saccharomyces cerevisiae under three different experimental conditions: in the whole lysate system, in a micrococcal-nuclease-treated lysate, and in a high-salt-treated lysate. The results indicate that translation of messenger is more resistant to edeine in the whole lysate than in the depleted lysates; these observations suggest that resistance to edeine is associated with the presence of endogenous mRNA. It is shown that 40S mutant subunits have a higher affinity for polysomal RNA than 40S wild-type subunits. Since the mRNA binding is inhibited by 7-methylguanosine 5'-monophosphate, the interaction between polysomal RNA and 40S ribosomes is specific for mRNA. The data demonstrate that in each of the depleted lysates, with edeine initially present, the formation of the 80S initiation complex is inhibited. However, edeine inhibition of [3H]methionine binding to 80S ribosomes is overcome completely in the mutant extract by preincubation of this lysate with polysomal RNA. The results indicate that the mutant may carry a specific change in a messenger-binding factor or in a ribosomal protein thereby permitting an increased stability of the messenger-ribosome complex which consequently results in an increased resistance of the mutant lysate to edeine.     

Codon-specific interaction of uncharged transfer-RNA with eukaryotic ribosomes.

Rat liver ribosomes bound [32P]tRNAPhe in both a codon-dependent and codon-independent manner. The codon-dependent binding was studied further by utilising the ability of the unchanged tRNAPhe to inhibit the poly(U)-directed binding of [3H]Phe-tRNA to ribosomes. At least part of the codon-dependent binding of uncharged tRNA appears to be to the ribosomal A-site.     

Crosslinking of N-acetyl-phenylalanyl [s4U]tRNAPhe to protein S10 in the ribosomal P site

In order to identify ribosomal components involved in the peptidyl-tRNA binding site on the ribosome, tRNAPhe molecules were prepared in which cytidine residues had been chemically converted into 4-thiouridine (S4U). This nucleoside is photoactive at 335 nm and able to form covalent bonds with nearby nucleophilic groups. The thiolated AcPhe-tRNAPhe was bound to the ribosomal P site in the presence of poly(U) as verified by puromycin reactivity. Direct irradiation of the AcPhe-[s4U]tRNAPhe poly(U) 70-S ribosome complex induced crosslinking of the tRNA molecule exclusively to 30-S subunits. Analysis of the covalent complex revealed that AcPhe-[s4U]tRNAPhe was specifically crosslinked to protein S10.     

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.     

Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system. J. Bacteriol. 92:197-203. 1966.-When tetracycline, an inhibitor of cell-free protein synthesis, was preincubated with each component of the Escherichia coli cell-free system, i.e., ribosomes, soluble ribonucleic acid (sRNA), polyuridylic acid (poly U), and S-100 (supernatant enzymes), only the ribosomal-bound antibiotic was inhibitory to the cell-free assay. Experiments designed to further localize the site of inhibition to either the 50S (Svedberg) or the 30S ribosomal subunit were not conclusive. Tritiated tetracycline (7-H(3)-tetracycline) was bound to isolated 50S ribosomes, and these were recombined with 30S subunits to form 70S ribosomes. When these ribosomes were dissociated and the subunits reisolated, the antibiotic was found with both the 50S and the 30S particles. The same results were observed when the tetracycline was initially bound to the 30S subunit.