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.     

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.     

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.     

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.     

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.     

Contacts of ribosomal proteins with tRNAPhe and 16S RNA in analogs of the 30S initiation complex

Direct RNA-protein contacts have been studied by means of ultraviolet-induced (254 nm) cross-links inside complexes of NAcPhe-tRNAPhe, Phe-tRNAPhe and deacylated tRNAPhe with poly(U)-charged 30S subunit of Escherichia coli ribosome. In the first two complexes tRNA directly contacts with the similar sets of proteins (S4, S5, S7, S9/S11; S6 and S8 are found only in the second complex). These sets are similar to that in the fMet-tRNAfMet X 30S X mRNA complex, evidencing similar disposition of tRNAs in these three complexes. 16S RNA contacts in free 30S subunit mainly with proteins S4, S7 and S9/S11. In both complexes, containing NAcPhe-tRNAPhe and Phe-tRNAPhe, 16S RNA contacts with essentially the same proteins (S4, S5, S7, S8, S9/S11, S10, S15, S16 and S17) and in the same ratio, evidencing similar conformation of 30S subunit in these two complexes. In the third complex deacylated tRNAPhe contacts with proteins S4, S5, S6, S8, S9/S11 and S15, 16S RNA-protein interaction differs from those in the first two complexes by a remarkable decrease of cross-linked proteins S8, and S9/S11 and by the appearance of a large amount of cross-linked proteins(s) S13/S14. Hence, this complex differs from the first two by conformation of 30S subunit and, probably, by disposition and/or conformation of tRNA.     

Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyl-tRNA molecular rulers.

A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe. Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.     

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.     

70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [3H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [3H]tRNA, or with [3H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the 'additional' site is not sensitive to the template. [3H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is preferred to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3'-end adenosine is essential for the affinity. The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.     

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.     

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.     

Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm

Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.     

Codon-anticodon interaction at the ribosomal P site improves the accuracy of the decoding process

The competing incorporation of [14C]Phe (cognate) and [3H]Leu (miscognate) into polypeptide material was measured. Kinetic analysis revealed that at the very beginning of protein synthesis the error fraction was 3 to 10 times larger than the "system-inherent" error, which is the error fraction found on the synthesized proteins. However, if a cognate tRNA, namely either deacylated tRNAPhe or AcPhe-tRNAPhe were prebound to the ribosomal P site, no enhanced error fraction was observed at the beginning. Furthermore, the initial rate of protein synthesis was increased by a factor of 10 to 50, regardless as to whether the ribosomes were precharged with tRNAPhe or AcPhe-tRNAPhe. In contrast, preincubation of ribosomes with non-cognate tRNA such as deacylated tRNALys had no effect. We conclude that codon-anticodon interaction at the P site facilitates the precise exposure of the adjacent codon at the ribosomal A site, thus contributing to the velocity and precision of the decoding process.     

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.     

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1. Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.     

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.     

Effect of the concentration ratio of bi- and monovalent ions on the functional activity of 30S ribosome subunits of Escherichia coli

Experiments on poly(U)-dependent binding of Phe-tRNAPhe to 30S subunits revealed the existence of a critical [Mg2+]/[NH4+] ratio in a medium (approximately 0.05-0.1) with respect to the binding capacity of subunits. If the ratio is greater than the critical one, 30S subunits undergo reversible inactivation even at the highest Mg2+ concentrations (up to 20 mM). The stronger is the deviation from the [Mg2+]/[NH4+] value = 0.05-0.1, the greater are both the rate and extent of such an inactivation. Two sites for tRNA in initially active 30S subunits have been shown to be inactivated in an interdependent way. On the other hand, a progressive decrease of [Mg2+]/[NH4+] ratio in a medium (from the value of 0.05 and lower) does not produce inactivation, but rather results in reduced affinity constants of Phe-tRNAPhe for active sites of 30S subunits.     

Different conformations of tRNA in the ribosomal P-site and A-site

Footprinting studies involving radioactively end-labelled tRNA species bound at either the ribosomal P- or A-site have yielded information that the tRNA's conformation is different in the two sites. Appropriate controls showed the relevance of using poly(U)-directed tRNAPhe binding in the P-site and Phe-tRNAPhe in the A-site. Digestion of the tRNA species was effected by RNases T1, T2 and cobra venom RNase. Experiments were performed with tRNAs 32P-labelled at either end to establish positions of primary cuts more confidently. In addition to the common protection of the aminoacyl-stem and anticodon-arm, footprinting experiments revealed striking differences in the accessibility of the T- and D-loops of tRNAs bound in the P- and A-sites. We observed a more open structure for the tRNA in the A-site. These results are consistent with a dynamic structure of tRNA during the translocation step of protein biosynthesis.     

Affinity modification of Escherichia coli ribosomes with the non-hydrolyzed GTP analog, gamma-[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP

Substituted gamma-amides of GTP viz. GTP gamma-[4-N-(2-chloro- and gamma-[4-N-(2-hydroxyethyl)-N-methylaminobenzyl]amide (CIRCH2NHpppG and OHRCH2NHpppG, resp.) were shown to be unhydrolisable GTP analogues in the EF-Tu-dependent GTP-ase reaction of ribosomes. The reactive analogue, CIRCH2NHpppG, was used for affinity labelling within the 70S ribosome.poly(U).tRNAPhe(P-site).Phe-tRNAPhe.EF-Tu.CIR[14C]CH2.NHpppG complex. Both 50S and 30S subunits were thus labelled but 50S subunit was modified considerably more than 30C subunit. Labelled were proteins L17, L21, S16, S21, and rRNA of both subunits, 23C rRNA within 50C subunit being labelled preferentially as compared with 50C proteins. No labelling of EF-Tu within the complex was detected.