Perturbation of the tRNA tertiary core differentially affects specific steps of the elongation cycle

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.     

Ribose 2'-hydroxyl groups in the 5' strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation

The coupled movement of tRNA-mRNA complex through the ribosome is a fundamental step during the protein elongation process. We demonstrate that the ribosome will translocate a P-site-bound tRNA(Met) with a break in the phosphodiester backbone between positions 17 and 18 in the D-loop. Crystallographic data showed that the acceptor arms of P- and E-site tRNA interact extensively with the ribosomal large subunit. Therefore, we used this fragmented P-site-bound tRNA(Met) to investigate the contributions of single 2'-hydroxyl groups in the 5' strand of the acceptor arm for translocation into the ribosomal E-site. EF-G-dependent translocation of the tRNAs was monitored using a toeprinting assay and a fluorescence-based rapid kinetic method. Surprisingly, our results show that none of the 2'-hydroxyl groups in the 5' strand of the acceptor arm of P-site-bound tRNA(Met) between positions 1-17 play a critical role during translocation. This suggests that either these 2'-hydroxyl groups are not important for translocation or they are redundant and the three-dimensional shape of the P-site tRNA is more important for translocation.     

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome

Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.     

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.     

Ribosomal crystallography: a flexible nucleotide anchoring tRNA translocation, facilitates peptide-bond formation, chirality discrimination and antibiotics synergism

The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA-end and P-site preference of initial tRNA. The interactions of protein L2 tail with the symmetry-related region periphery explain its conservation and its contributions to nascent chain elongation.     

Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA^Leu recognition sets and their evolution, the recognition of tRNA^Leu by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8–A14, G18–U55 and G19–C56; and the orientation of the variable arm are critical elements for tRNA^Leu aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.     

EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome.

Translocation, catalyzed by elongation factor EF-G, is the precise movement of the tRNA-mRNA complex within the ribosome following peptide bond formation. Here we examine the structural requirement for A- and P-site tRNAs in EF-G-catalyzed translocation by substituting anticodon stem-loop (ASL) analogs for the respective tRNAs. Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting. Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA-dependent fashion. The requirement for an A-site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop. Translocation of the ASL is both EF-G- and GTP-dependent, and is inhibited by the translocational inhibitor thiostrepton. These findings show that the D, T and acceptor stem regions of A-site tRNA are not essential for EF-G-dependent translocation. In contrast, no translocation occurs if the P-site tRNA is substituted with an ASL, indicating that other elements of P-site tRNA structure are required for translocation. We also tested the effect of increasing the A-site ASL stem length from 4 to 33 bp on translocation from A to P site. Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon-D arm axis. This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon-D arm.     

Mechanism of translation based on intersubunit complementarities of ribosomal RNAs and tRNAs

A universal rule is found about nucleotide sequence complementarities between the regions 2653-2666 in the GTPase-binding site of 23S rRNA and 1064-1077 of 16S rRNA as well as between the region 1103-1107 of 16S rRNA and GUUCG (or GUUCA) of tRNAs. This rule holds for all species in the living kingdoms except for two protista mitochondrial rRNAs of Trypanosoma brucei and Plasmodium falciparum. We found that quite similar relationships for the two species hold under the assumption presented in the present paper. The complementarity between T-loop of tRNA and the region 1103-1107 of 16S rRNA suggests that the first interaction of a ribosome with aminoacyl-tRNAEF-TuGTP ternary complex or EF-GGDP complex could occur at the region 1103-1107 of 16S rRNA with the T-loop-D-loop contact region of the ternary complex or the domain IV-V bridge region of the EF-GGDP complex. The second interaction should occur between the A-site codon and the anticodon loop or between the anticodon stem/loop of A-site tRNA and the tip of domain IV of EF-G. The above stepwise interactions would facilitate the collision of the region 1064-1077 of 16S rRNA with the region around A2660 at the alpha-sarcin/ricin loop of 23S rRNA. In this way, the universal rule is capable of explaining how spectinomycin-binding region of 16S rRNA takes part in translocation, how GTPases such as EF-Tu and EF-G can be introduced into their binding site on the large subunit ribosome in proper orientation efficiently and also how driving forces for tRNA movement are produced in translocation and codon recognition. The analysis of T-loops of all tRNAs also presents an evolutionary trend from a random and seemingly primitive sequence, as defined to be Y type, to the most developed structure, such as either 5G7 or 5A7 types in the present definition.     

Translational properties of mHNA, a messenger RNA containing anhydrohexitol nucleotides

Short messenger RNAs (mRNAs) with hexitol residues in two codons were constructed and their properties were studied in an Escherichia coli in vitro translation system. The replacement of the natural ribonucleotides of mRNA in the AUG start codon and the UUC second codon by hexitol nucleotides did not influence the main steps of translation, as indicated by the same level of binding of mRNA with or without hexitol residues under P-site conditions, and the same yield of tRNA binding to the P- and A-sites. Moreover, both peptide formation and translocation took place on mRNAs with hexitol residues. The presence of an A-type messenger hexitol nucleic acid (mHNA)-transfer RNA (tRNA) duplex is important for efficient translation and the 2'-OH function in mRNA is not necessary for binding and movement through the ribosome. Groove shape recognition of the codon-anticodon complex, more than hydrogen-bond interactions of ribose residues in mRNA, is an important factor for correct translation.     

Reverse translocation of tRNA in the ribosome

A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation.     

Crosslinking of translation factor EF-G to proteins of the bacterial ribosome before and after translocation

Elongation factor G (EF-G) promotes the translocation of tRNA and mRNA in the central cavity of the ribosome following the addition of each amino acid residue to a growing polypeptide chain. tRNA/mRNA translocation is coupled to GTP hydrolysis, catalyzed by EF-G and activated by the ribosome. In this study we probed EF-G interactions with ribosomal proteins (r-proteins) of the bacterial ribosome, by using a combination of chemical crosslinking, immunoblotting and mass spectroscopy analyses. We identified three bacterial r-proteins (L7/L12, S12 and L6) crosslinked to specific residues of EF-G in three of its domains (G', 3 and 5, respectively). EF-G crosslinks to L7/L12 and S12 were indistinguishable when EF-G was trapped on the ribosome before or after tRNA/mRNA translocation had occurred, whereas a crosslink between EF-G and L6 formed with greater efficiency before translocation had occurred. EF-G crosslinked to L7/L12 was capable of catalyzing multiple rounds of GTP hydrolysis, whereas EF-G crosslinked to S12 was inactive in GTP hydrolysis. These results imply that during the GTP hydrolytic cycle EF-G must detach from S12 within the central cavity of the ribosome, while EF-G can remain associated with L7/L12 located on one of the peripheral stalks of the ribosome. This mechanism may ensure that a single GTP molecule is hydrolyzed for each tRNA/mRNA translocation event.     

The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.     

Insights into the molecular determinants of EF-G catalyzed translocation

Translocation, the directional movement of transfer RNA (tRNA) and messenger RNA (mRNA) substrates on the ribosome during protein synthesis, is regulated by dynamic processes intrinsic to the translating particle. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, in combination with site-directed mutagenesis of the ribosome and tRNA substrates, we show that peptidyl-tRNA within the aminoacyl site of the bacterial pretranslocation complex can adopt distinct hybrid tRNA configurations resulting from uncoupled motions of the 3'-CCA terminus and the tRNA body. As expected for an on-path translocation intermediate, the hybrid configuration where both the 3'-CCA end and body of peptidyl-tRNA have moved in the direction of translocation exhibits dramatically enhanced puromycin reactivity, an increase in the rate at which EF-G engages the ribosome, and accelerated rates of translocation. These findings provide compelling evidence that the substrate for EF-G catalyzed translocation is an intermediate wherein the bodies of both tRNA substrates adopt hybrid positions within the translating ribosome.     

Coupling of GTP hydrolysis by elongation factor G to translocation and factor recycling on the ribosome

The translocation step of elongation entails the coordinated movement of tRNA and mRNA on the ribosome. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis, which affects both translocation and turnover of EF-G. Both reactions are much slower (50-100-fold) when GTP is replaced with non-hydrolyzable GTP analogues or GDP, indicating that the reaction rates are determined by conformational transitions induced by GTP hydrolysis. Compared to the rate of uncatalyzed, spontaneous translocation, ribosome binding of EF-G with any guanine nucleotide reduces the free energy of activation by about 18 kJ/mol, whereas GTP hydrolysis contributes another 10 kJ/mol. The acceleration by GTP hydrolysis is due to large decrease in activation enthalpy by about 30 kJ/mol, compared to the reaction with GTP analogues or GDP, whereas the activation entropy becomes unfavorable and is lowered by about 20 kJ/mol (37 degrees C). The data suggest that GTP hydrolysis induces, by a conformational change of EF-G, a rapid conformational rearrangement of the ribosome ("unlocking") which determines the rates of both tRNA-mRNA translocation and recycling of the factor.     

Effect of mutations in the nucleocapsid protein (NCp7) upon Pr160(gag-pol) and tRNA(Lys) incorporation into human immunodeficiency virus type 1.

COS-7 cells were transfected with DNAs containing mutations in the NCp7 sequences of human immunodeficiency virus. Selective incorporation into the virus of tRNA(Lys) was measured by two-dimensional polyacrylamide gel electrophoresis, and Pr160(gag-pol) incorporation into the virus was detected in Western blots of viral protein. Mutations tested included cysteine and histidine mutations in either of the Cys-His boxes, as well as mutations in the N- and C-terminal flanking regions and in the linker region between the two Cys-His boxes. Of 10 mutations tested, only 2 inhibited tRNA(Lys) incorporation: a P31L mutation in the linker region and a deletion which removed both Cys-His boxes and the linker region (deltaK14-T50). The P31L mutation prevents the incorporation of Pr160(gag-pol) into the virus. Cotransfection of COS cells with both P31L DNA and a plasmid coding only for unprocessed Pr160(gag-pol) resulted in the viral incorporation of Pr160(gag-pol) and the rescue of selective packaging of tRNA(Lys) into the virion. In the deltaK14-T50 mutant, Pr160(gag-pol) is incorporated into the virus. Selective tRNA(Lys) packaging is not rescued by cotransfection with a plasmid coding for Pr160(gag-pol) but is rescued by cotransfection with DNA coding for wild-type Pr55(gag). Since Pr55(gag) does not by itself selectively package tRNA(Lys), the deltaK14-T50 mutation may be affecting tRNA(Lys) binding to a cytoplasmic Pr55(gag)/Pr160(gag-pol) complex.     

GTP hydrolysis by EF‐G synchronizes tRNA movement on small and large ribosomal subunits

Elongation factor G (EF‐G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF‐G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF‐G mutants and translocation‐specific antibiotics to investigate timing and energetics of translocation. We show that EF‐G–GTP facilitates synchronous movements of peptidyl‐tRNA on the two subunits into an early post‐translocation state, which resembles a chimeric state identified by structural studies. EF‐G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF‐G. Our results reveal two distinct modes for utilizing the energy of EF‐G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit.     

EFG-independent translocation of the mRNA:tRNA complex is promoted by modification of the ribosome with thiol-specific reagents

Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.     

Kinetically competent intermediates in the translocation step of protein synthesis

Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.     

Spontaneous reverse movement of mRNA-bound tRNA through the ribosome

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.     

Structural dynamic aspects of protein synthesis on ribosomes

Three types of conformational changes in the translating ribosome are considered: (1) intersubunit movement (ribosome unlocking) during translocation; (2) L7/L12 stalk mobility affected by elongation factors; (3) change of tRNA residue during its transition from the A-site to the P-site. Relevant experimental data are reviewed.