Distortion of tRNA upon near-cognate codon recognition on the ribosome

The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or near-cognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre- to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.     

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.     

Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex

The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex. The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket. These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.     

SmpB triggers GTP hydrolysis of elongation factor Tu on ribosomes by compensating for the lack of codon-anticodon interaction during trans-translation initiation

Bacterial tmRNA rescues ribosomes that stall because of defective mRNAs via the trans-translation process. Although entry of the charged transfer messenger RNA (tmRNA) into the ribosome proceeded in the absence of elongation factor (EF-Tu) and in the presence of EF-Tu and the antibiotic kirromycin, evidence was found for the involvement of EF-Tu in trans-translation initiation. The polyalanine synthesis system attained by using a tmRNA variant consisting of only the tRNA-like domain revealed that it was completely dependent on the presence of SmpB and greatly enhanced by EF-Tu and EF-G. Actually, ribosome-dependent GTPase activity of EF-Tu was stimulated by the addition of SmpB and tmRNA but independently of template mRNA, demonstrating that SmpB compensates for the lack of codon-anticodon interaction during the first step of the trans-translation initiation. Based on these results, we suggest that SmpB structurally mimics the anticodon arm of tRNA and elicits GTP hydrolysis of EF-Tu upon tmRNA accommodation in the A site of the ribosome.     

Thiostrepton inhibition of tRNA delivery to the ribosome

Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.     

Kinetic basis for global loss of fidelity arising from mismatches in the P-site codon:anticodon helix

Faithful decoding of the genetic information by the ribosome relies on kinetically driven mechanisms that promote selection of cognate substrates during elongation. Recently, we have shown that in addition to these kinetically driven mechanisms, the ribosome possesses a post peptidyl transfer quality control system that retrospectively monitors the codon–anticodon interaction in the P site, triggering substantial losses in the specificity of the A site during subsequent tRNA and RF selection when a mistake has occurred. Here, we report a detailed kinetic analysis of tRNA selection in the context of a mismatched P-site codon:anticodon interaction. We observe pleiotropic effects of a P-site mismatch on tRNA selection, such that near-cognate tRNA is processed by the ribosome almost as efficiently as cognate. In particular, after a miscoding event, near-cognate codon–anticodon complexes are stabilized on the ribosome to an extent similar to that observed for cognate ones. Moreover, the two observed forward rates of GTPase activation and accommodation are greatly accelerated (∼10-fold) for near-cognate tRNAs. Because the ensemble of effects of a mismatched P site on substrate selection were found to be different from those reported for other ribosomal perturbations and miscoding agents, we propose that the structural integrity of the mRNA–tRNA helix in the P site provides a distinct molecular switch that dictates the specificity of the A site.     

Distinct functional classes of ram mutations in 16S rRNA

During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EF-Tu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed “accommodation”) or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.     

Possible evolution of factors involved in protein biosynthesis.

The elongation factors of protein biosynthesis are well preserved through out evolution. They catalyze the elongation phase of protein biosynthesis, where on the ribosome amino acids are added one at a time to a growing peptide according to the genetic information transcribed into mRNA. Elongation factor Tu (EF-Tu) provides the binding of aminoacylated tRNA to the ribosome and protects the aminoester bond against hydrolysis until a correct match between the codon on mRNA and the anticodon on tRNA can be achieved. Elongation factor G (EF-G) supports the translocation of tRNAs and of mRNA on the ribosome so that a new codon can be exposed for decoding. Both these factors are GTP binding proteins, and as such exist in an active form with GTP and an inactive form with GDP bound to the nucleotide binding domain. Elongation factor Ts (EF-Ts) will catalyze the exchange of nucleotide on EF-Tu. This review describes structural work on EF-Tu performed in our laboratory over the last eight years. The structural results provide a rather complete picture of the major structural forms of EF-Tu, including the so called ternary complex of aa-tRNA:EF-Tu:GTP. The structural comparison of this ternary complex with the structure of EF-G:GDP displays an unexpected macromolecular mimicry, where three domains of EF-G mimick the shape of the tRNA in the ternary complex. This observation has initiated much speculation on the evolution of all factors involved in protein synthesis, as well as on the details of the ribosomal function in one part of elongation.     

An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon–anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB''s affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.     

Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome

Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.     

The central role of protein S12 in organizing the structure of the decoding site of the ribosome

The ribosome decodes mRNA by monitoring the geometry of codon–anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.     

Molecular dynamics of EF-G during translocation

Elongation factor G (EF‐G) plays a crucial role in two stages of mRNA‐(tRNA)₂ translocation. First, EF‐G?GTP enters the pre‐translocational ribosome in its intersubunit‐rotated state, with tRNAs in their hybrid (P/E and A/P) positions. Second, a conformational change in EF‐G's Domain IV induced by GTP hydrolysis disengages the mRNA‐anticodon stem‐loops of the tRNAs from the decoding center to advance relative to the small subunit when the ribosome undergoes a backward inter‐subunit rotation. These events take place as EF‐G undergoes a series of large conformational changes as visualized by cryo‐EM and X‐ray studies. The number and variety of these structures leave open many questions on how these different configurations form during the dynamic translocation process. To understand the molecular mechanism of translocation, we examined the molecular motions of EF‐G in solution by means of molecular dynamics simulations. Our results show: (1) rotations of the super‐domain formed by Domains III–V with respect to the super‐domain formed by I–II, and rotations of Domain IV with respect to Domain III; (2) flexible conformations of both 503‐ and 575‐loops; (3) large conformational variability in the bound form caused by the interaction between Domain V and the GTPase‐associated center; (4) after GTP hydrolysis, the Switch I region seems to be instrumental for effecting the conformational change at the end of Domain IV implicated in the disengagement of the codon‐anticodon helix from the decoding center. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Recognition and selection of tRNA in translation

Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. The stepwise movement of aa-tRNA from EF-Tu into the ribosomal A site entails a number of intermediates. The ribosome recognizes aa-tRNA through shape discrimination of the codon-anticodon duplex and regulates the rates of GTP hydrolysis by EF-Tu and aa-tRNA accommodation in the A site by an induced fit mechanism. Recent results of kinetic measurements, ribosome crystallography, single molecule FRET measurements, and cryo-electron microscopy suggest the mechanism of tRNA recognition and selection.     

Elongation factors on the ribosome

The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.     

Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu

The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not peptidyltransferase—the essential catalytic activity of the large ribosomal subunit—also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA•EF-Tu•GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit.     

Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome.

The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.     

Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome

Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis. The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood. It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis. However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified. Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10(6)-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly. These results show that His84 plays a key role in the chemical step of GTP hydrolysis. Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPgammaS, showed no dependence on pH, indicating that His84 does not act as a general base. We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the gamma-phosphate group of GTP.     

Multiple stages in codon-anticodon recognition: double-trigger mechanisms and geometric constraints

Thirty years of kinetic studies on tRNA selection in the elongation cycle are reviewed, and confronted with results derived from various sources, including structural studies on the ribosome, genetic observations on ribosome and EF-Tu accuracy mutants, and codon-specific elongation rates. A coherent framework is proposed, which gives meaning to many puzzling effects. Ribosomal accuracy would be governed by a "double-trigger" principle, according to which the ribosome uses energy in the forward direction to create new configurations for tRNA selection, and energy in the backward direction to regain its initial configuration, in particular after a premature dissociation event. The conformation energy would come in part, in Hopfield's mode, from GTP cleavage on the ternary complex (TC). The reset energy would be provided in part, in the author's mode, from GTP cleavage on a binary EF-Tu.GTP complex (BC). There would be several paths for amino acid incorporation. The path of highest accuracy would involve TC binding followed by BC binding, followed either by GTP hydrolysis on the TC, or by TC dissociation and GTP hydrolysis on the BC. Codon-anticodon recognition would occur in at least three kinetically and geometrically distinct stages. In a first stage, there would be a very rapid sorting of the TCs with unstrained anticodons contacting a loosely held mRNA. This stage ends with the anchoring of the codon-anticodon complex by a cluster of three nucleotides of 16S RNA. The second stage would be the most discriminative one. It would operate on the 5 ms time scale and terminate with GTP cleavage on the TC. The third stage would provide a last, crude selection involving "naked" aa-tRNA, partially held back by steric hindrance. Streptomycin and most EF-Tu mutants as well as high accuracy ribosomal mutants would produce specific alterations at stage 2, which are mapped on the stage 2 kinetic mechanism. The ram ribosomal ambiguity mutants, and anticodon position 37 modifications could be markers of stages 1 and 3 selection. Dissociation events at stage 2 or stage 3, when they are not immediately followed by reset events create a leaky state favorable to shortcut incorporation events. These events are equivalent to an "error-prone codon-anticodon mismatch repair". From the recent evidence on ribosome structure, it is conjectured that the L7/L12 flexible stalk of the large ribosome subunit acts as a proofreading gate, and that the alternation of its GTPase activation center between "TCase" competence and "BCase" competence is a main factor in the control of accuracy.     

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and P_i release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.     

Functions and interplay of the tRNA-binding sites of the ribosome

The ribosome translates the genetic information of an mRNA molecule into a sequence of amino acids. The ribosome utilizes tRNAs to connect elements of the RNA and protein worlds during protein synthesis, i.e. an anticodon as a unit of genetic information with the corresponding amino acid as a building unit of proteins. Three tRNA-binding sites are located on the ribosome, termed the A, P and E sites. In recent years the tRNA-binding sites have been localized on the ribosome by three different techniques, small-angle neutron scattering, cryo-electron microscopy and X-ray analyses of 70 S crystals. These high-resolution glimpses into various ribosomal states together with a large body of biochemical data reveal an intricate interplay between the tRNAs and the three ribosomal binding sites, providing an explanation for the remarkable features of the ribosome, such as the ability to select the correct ternary complex aminoacyl-tRNA.EF-Tu.GTP out of more than 40 extremely similar tRNA complexes, the precise movement of the tRNA(2).mRNA complex during translocation and the maintenance of the reading frame.