Extension of Watson's Model for the Elongation Cycle of Protein Biosynthesis

Abstract

The scheme for the elongation cycle of protein biosynthesis is proposed based on modern quantitative data on the interactions of mRNA and different functional forms of tRNA with 70S ribosomes and their 30S and 50S subunits. This scheme takes into account recently discovered third ribosomal (E) site with presumable exit function. The E site is introduced into 70S ribosome by its 50S subunit, the codon-anticodon interaction does not take place at the E site, and the affinity of tRNA for the E site is considerably lower than that for the P site. On the other hand, the P and A sites are located mainly on a 30S subunit, the codon-anticodon interactions being realized on both these sites. An mRNA molecule is placed exclusively on a 30S subunit where it makes U-turn. The proposed scheme does not contradict to any data but includes all main postulates of the initial Watson's model (J. D. Watson, Bull Soc. Chim. Biol. 46, 1399 (1964), and is considered as a natural extension of the later according to modern experimental data.     

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.     

Codon-anticodon interaction at the P site is a prerequisite for tRNA interaction with the small ribosomal subunit

The arrival of high resolution crystal structures for the ribosomal subunits opens a new phase of molecular analysis and asks for corresponding analyses of ribosomal function. Here we apply the phosphorothioate technique to dissect tRNA interactions with the ribosome. We demonstrate that a tRNA bound to the P site of non-programmed 70 S ribosomes contacts predominantly the 50 S, as opposed to the 30 S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30 S binding. Protection patterns of tRNAs bound to isolated subunits and programmed 70 S ribosomes were compared. The results suggest the presence of a movable domain in the large ribosomal subunit that carries tRNA and reveal that only approximately 15% of a tRNA, namely residues 30 +/- 1 to 43 +/- 1, contact the 30 S subunit of programmed 70 S ribosomes, whereas the remaining 85% make contact with the 50 S subunit. Identical protection patterns of two distinct elongator tRNAs at the P site were identified as tRNA species-independent phosphate backbone contacts. The sites of protection correlate nicely with the predicted ribosomal-tRNA contacts deduced from a 5.5-A crystal structure of a programmed 70 S ribosome, thus refining which ribosomal components are critical for tRNA fixation at the P site.     

Effect of modification of tRNA nucleotide 37 on the tRNA interaction with the A and P sites of the Escherichia coli 70S ribosome

The modified nucleotide 3′ of the tRNA anticodon is an important structural element that regulates the codon-anticodon interaction in the ribosome by stacking with codon-anticodon bases. The presence and identity (pyrimidine, purine, or modified purine) of this nucleotide significantly affects the energy of stacking in the A and P sites of the ribosome. Modification of nucleotide 37 does not contribute to stacking in the A site of the 70S ribosome, while its effect is substantial in the P site. The enthalpies of tRNA interactions with the A and P sites in the ribosome are similar and considerably lower than the enthalpy of the interactions of two tRNAs with the cognate anticodons in solution, suggesting that the ribosome contributes to the enthalpy-related portion of the free energy of tRNA binding by directly forming additional interactions with tRNA or by indirectly stabilizing the conformation of the codon-anticodon complex. In addition to stacking, tRNA binding in the A and P sites is further stabilized by interactions that involve magnesium ions. The number of ions involved in the formation of the tRNA-ribosome complex depends on the identity of tRNA nucleotide 37.     

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.     

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.     

Two tRNA-binding sites in addition to A and P sites on eukaryotic ribosomes.

The interaction of tRNA with 80 S ribosomes from rabbit liver was studied using biochemical as well as fluorescence techniques. Besides the canonical A and P sites, two additional sites were found which specifically bind deacylated tRNA. One of the sites is analogous to the E site of prokaryotic ribosomes, in that binding of tRNA is labile, does not depend on codon-anticodon interaction, does not protect the anticodon loop from solvent access, and requires the presence of the 3'-terminal adenosine of the tRNA. In contrast, the stability of the tRNA complex with the second site (S site) is high. tRNA binding to the S site is also codon-independent; nevertheless, the anticodon loop is shielded from solvent access. Removal of the 3'-terminal adenosine decreases the affinity of tRNA(Phe) for the S site approximately 50-fold. tRNA(Phe) is retained at the S site during translocation and through poly(Phe) synthesis. Thus, the S site does not seem to be an intermediate site for the tRNA during the elongation cycle. Rather, the tRNA bound to the S site may allosterically modulate the function of the ribosome.     

Adjacent codon-anticodon interactions of both tRNAs present at the ribosomal A and P or P and E sites

A labeled tRNA present at the A, P or E site can be partially chased from the ribosome, a cognate nonlabeled tRNA as chasing substrate being 3-12-times more efficient than non-cognate tRNA at a molar ratio tRNA: 70 S = 10:1. These findings indicate that a tRNA bound to a programmed ribosome undergoes codon-anticodon interaction at all three sites (A, P and E site). Furthermore, both labeled tRNA present on the ribosome can be chased more effectively with cognate than with non-cognate substrate at the same time. This finding provides strong evidence that both tRNAs present on the ribosome exhibit simultaneous codon-anticodon interaction. This is valid for both the pretranslocational state (Ac[3H]Lys-tRNALys in the A and [14C]tRNALys in the P site) as well as the posttranslocational state (Ac[3H]Lys-tRNALys in the P and [14C]tRNALys in the E site).     

How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography.

Two tRNA molecules at the ribosomal A- and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations. In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA. A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration. In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit. The translocation step is essentially a rotational movement of the tRNA from the A- to the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit. The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the A- to the P-site in the 3' to 5'-direction. These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit. The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with A- or P-site tRNA, favour the latter situation. This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome.     

Quantitative analysis of deoxynucleotide substitutions in the codon-anticodon helix

The role of 2' hydroxyl groups in the codon-anticodon helix was evaluated by introducing single deoxynucleotides into each of the six positions in the helix and measuring the affinity of tRNA to either the A site or the P site of Escherichia coli 70S ribosomes. In perfect agreement with the X-ray structure of the Thermus thermophilus 30S subunit, A site binding was weaker in five of the six positions but P site binding was unaffected. Since the addition of paromomycin restores A site binding, it appears that the deoxynucleotide substituted complexes are impaired in their ability to promote the ribosomal conformational change that accompanies tRNA binding.     

Destabilization of codon-anticodon interaction in the ribosomal exit site

The affinities of the exit (E) site of poly(U) or poly(A)-programmed Escherichia coli ribosomes for the respective cognate tRNA and a number of non-cognate tRNAs were determined by equilibrium titrations. Among the non-cognate tRNAs, the binding constants vary up to about tenfold (10(6) to 10(7) M-1 at 20 mM-Mg2+) or 50-fold (10 mM-Mg2+), indicating that codon-independent binding is modulated to a considerable extent by structural elements of the tRNA molecules other than the anticodon. Codon-anticodon interaction stabilizes tRNA binding in the E site approximately fourfold (20 mM-Mg2+) or 20-fold (10 mM-Mg2+), corresponding to delta G degree values of -3 and -7 kJ/mol (0.7 and 1.7 kcal/mol), respectively. Thus, the energetic contribution of codon-anticodon interaction to tRNA binding in the E site appears rather small, particularly in comparison to the large effects on the binding in A and P sites and to the binding of complementary oligonucleotides or of tRNAs with complementary anticodons. This result argues against a role of the E site-bound tRNA in the fixation of the mRNA on the ribosome. In contrast, we propose that the role of the E site is to facilitate the release of the discharged tRNA during translocation by providing an intermediate, labile binding site for the tRNA leaving the P site. The lowering of both affinity and stability of tRNA binding accompanying the transfer of the tRNA from the P site to the E site is predominantly due to the labilization of the codon-anticodon interaction.     

How initiation factors tune the rate of initiation of protein synthesis in bacteria

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)-programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell-free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis-Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3-free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.     

Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.     

The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.     

The side-by-side model of two tRNA molecules allowing the alpha-helical conformation of the nascent polypeptide during the ribosomal transpeptidation.

Lim and Spirin [25] proposed a preferable conformation of the nascent peptide during the ribosomal transpeptidation. Spirin and Lim [26] excluded the possibilities of the side-by-side model proposed by Johnson et al [13] and the three-tRNA binding model (A, P and E sites) of Rheinberger and Nierhaus [3]. However, a slight conformational change at the 3' end regions of both A and P site tRNA molecules can enable the three different tRNA binding models to converge. With a modification of the angles of the ribose rings of both anticodon and mRNA this model can also be related to the model of Sundaralingam et al [19]. In this model of E coli rRNA the 3' end sequence ACCA76 or GCCA76 of P site tRNA is base-paired to UGGU810 of 23S rRNA, while the ACC75 or GCC75 of A site tRNA are base-paired to GGU1621 23S rRNA. The conformation of the A76 of A site tRNA is necessarily different from that of P site tRNA, at least during the course of the transpeptidation. The A76 of A site tRNA overlaps the binding region of puromycin. The C1400 of 16S rRNA in this model is located at a distance of 4 A from the 5' end of the anticodon of P site tRNA [14] and 17 A from the 5' end of the anticodon of A site tRNA [15]. It is also shown that a considerable but reasonable modification in the conformation of the anticodon loops could lead to accommodation of three deacylated tRNA(Phe) molecules at a time on 70S ribosome in the presence of poly(U) as observed experimentally [6]. A sterochemical explanation for the negatively-linked allosteric interactions between the A and E sites is also shown in the present model.     

Intact aminoacyl-tRNA is required to trigger GTP hydrolysis by elongation factor Tu on the ribosome

GTP hydrolysis by elongation factor Tu (EF-Tu) on the ribosome is induced by codon recognition. The mechanism by which a signal is transmitted from the site of codon-anticodon interaction in the decoding center of the 30S ribosomal subunit to the site of EF-Tu binding on the 50S subunit is not known. Here we examine the role of the tRNA in this process. We have used two RNA fragments, one which contains the anticodon and D hairpin domains (ACD oligomer) derived from tRNA(Phe) and the second which comprises the acceptor stem and T hairpin domains derived from tRNA(Ala) (AST oligomer) that aminoacylates with alanine and forms a ternary complex with EF-Tu. GTP. While the ACD oligomer and the ternary complex containing the Ala-AST oligomer interact with the 30S and 50S A site, respectively, no rapid GTP hydrolysis was observed when both were bound simultaneously. The presence of paromomycin, an aminoglycoside antibiotic that binds to the decoding site and stabilizes codon-anticodon interaction in unfavorable coding situations, did not increase the rate of GTP hydrolysis. These results suggest that codon recognition as such is not sufficient for GTPase activation and that an intact tRNA molecule is required for transmitting the signal created by codon recognition to EF-Tu.     

Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons

Mutant tRNAs containing an extra nucleotide in the anticodon loop are known to suppress +1 frameshift mutations, but in no case has the molecular mechanism been clarified. It has been proposed that the expanded anticodon pairs with a complementary mRNA sequence (the frameshift sequence) in the A site, and this quadruplet "codon-anticodon" helix is translocated to the P site to restore the correct reading frame. Here, we analyze the ability of tRNA analogs containing expanded anticodons to recognize and position mRNA in ribosomal complexes in vitro. In all cases tested, 8 nt anticodon loops position the 3' three-quarters of the frameshift sequence in the P site, indicating that the 5' bases of the expanded anticodon (nucleotides 33.5, 34, and 35) pair with mRNA in the P site. We also provide evidence that four base-pairs can form between the P-site tRNA and mRNA, and the fourth base-pair involves nucleotide 36 of the tRNA and lies toward (or in) the 30 S E site. In the A site, tRNA analogs with the expanded anticodon ACCG are able to recognize either CGG or GGU. These data imply a flexibility of the expanded anticodon in the A site. Recognition of the 5' three-quarters of the frameshift sequence in the A site and subsequent translocation of the expanded anticodon to the P site results in movement of mRNA by four nucleotides, explaining how these tRNAs can change the mRNA register in the ribosome to restore the correct reading frame.     

Locking and unlocking of ribosomal motions

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.