单分子荧光共振能量转移技术在核糖体移位研究中的进展

核糖体是蛋白质的"合成工厂",也是临床上多种抗菌药物的作用靶点,因此,深入理解细菌核糖体的蛋白质翻译机制意义重大.蛋白质翻译是通过多步骤相互协调、多组分精细配合来实现高保真和精确调控.核糖体在mRNA上的移位作为翻译过程中最重要的事件之一,需要核糖体大规模的构象重排以及tRNA2-mRNA沿着核糖体的精确移动.在细菌中,移位是由延伸因子EF-G催化GTP水解来驱动的.近年来,单分子荧光共振能量技术(smFRET)的发展使得人们可以探究单个tRNA分子移位的动力学过程并实时观测核糖体的构象变化.本文首先介绍了smFRET技术的原理及特点,对其在核糖体结构动态及tRNA移位研究中的应用进行了较为系统的总结,并对其应用前景进行了展望.     

Dynamics of Recognition between tRNA and elongation factor Tu

Elongation factor Tu (EF-Tu) binds to all standard aminoacyl transfer RNAs (aa-tRNAs) and transports them to the ribosome while protecting the ester linkage between the tRNA and its cognate amino acid. We use molecular dynamics simulations to investigate the dynamics of the EF-Tu·guanosine 5′-triphosphate·aa-tRNA^Cys complex and the roles played by Mg²⁺ ions and modified nucleosides on the free energy of protein·RNA binding. Individual modified nucleosides have pronounced effects on the structural dynamics of tRNA and the EF-Tu·Cys-tRNA^Cys interface. Combined energetic and evolutionary analyses identify the coevolution of residues in EF-Tu and aa-tRNAs at the binding interface. Highly conserved EF-Tu residues are responsible for both attracting aa-tRNAs as well as providing nearby nonbonded repulsive energies that help fine-tune molecular attraction at the binding interface. In addition to the 3′ CCA end, highly conserved tRNA nucleotides G1, G52, G53, and U54 contribute significantly to EF-Tu binding energies. Modification of U54 to thymine affects the structure of the tRNA common loop resulting in a change in binding interface contacts. In addition, other nucleotides, conserved within certain tRNA specificities, may be responsible for tuning aa-tRNA binding to EF-Tu. The trend in EF-Tu·Cys-tRNA^Cys binding energies observed as the result of mutating the tRNA agrees with experimental observation. We also predict variations in binding free energies upon misacylation of tRNA^Cys with d-cysteine or O-phosphoserine and upon changing the protonation state of l-cysteine. Principal components analysis in each case reveals changes in the communication network across the protein·tRNA interface and is the basis for the entropy calculations.     

The ribosome‐bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex

Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet‐tRNA^fMet to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet‐tRNA^fMet to the ribosome in a ternary complex, IF2·GTP·fMet‐tRNA^fMet. By using rapid kinetic techniques, we show here that binding of IF2·GTP to the 30S ribosomal subunit precedes and is independent of fMet‐tRNA^fMet binding. The ternary complex formed in solution by IF2·GTP and fMet‐tRNA is unstable and dissociates before IF2·GTP and, subsequently, fMet‐tRNA^fMet bind to the 30S subunit. Ribosome‐bound IF2 might accelerate the recruitment of fMet‐tRNA^fMet to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF‐Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.     

Structural modelling of the complex of leucyl-tRNA synthetase and mis-aminoacylated tRNA

To assure fidelity of translation, class Ia aminoacyl-tRNA synthetases (aaRSs) edit mis-aminoacylated tRNAs. Mis-attached amino acids and structural water molecules are not included simultaneously in the current crystal structures of the aaRS•tRNA complexes, where the 3′-ends (adenine 76; A76) are bound to the editing sites. A structural model of the completely solvated leucyl-tRNA synthetase complexed with valyl-tRNA^Leu was constructed by exploiting molecular dynamics simulations modified for the present modelling. The results showed that the ribose conformation of A76 is distinct from those observed in the above-mentioned crystal structures, which could be derived from structural constraints in a sandwiched manner induced by the mis-attached valine and tRNA^Leu.     

Classical molecular dynamics simulation of seryl tRNA synthetase and threonyl tRNA synthetase bound with tRNA and aminoacyl adenylate

Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (^ttSerRS) bound with ^tttRNA^Ser and dimeric ThrRS from Escherichia coli (^ecThrRS) bound with ^ectRNA^Thr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. These interactions help in the development of catalytically effective organization of the active site. The A76 end of the ^tttRNA^Ser exhibits fast dynamics in free State, which is significantly slowed down within the active site bound with adenylate. The loops change their conformation via multimodal dynamics (a slow diffusive mode of nanosecond time scale and fast librational mode of dynamics in picosecond time scale). The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg²⁺ ions around tRNA and dynamically slow bound water are other common features of both aaRSs. The presence of dynamically rigid Zinc ion coordination sphere and bipartite mode of recognition of ^ectRNA^Thr are observed.     

Model of the structure of the eukaryotic ribosomal translation termination complex

Models of the atomic structure of the eukaryotic translation termination complex containing mRNA, P-site tRNA^Phe, human class 1 release factor eRF1, and 80S ribosome, were constructed by computational modeling. The modeling was based on the assumed structural-functional similarity between the tRNA and eFR1 molecules in the ribosomal A site. The known atomic structure of the 70S ribosome complexed with mRNA as well as the P-and A-site tRNAs^Phe was used as a structural template for the modeling. The eRF1 molecule bound in the A site undergoes substantial conformational changes so that the mutual configuration of the N and M domains matches the overall tRNA shape. Two models of eRF1 binding to mRNA at the A site in the presence of P-site tRNA^Phe were generated. A characteristic of these models is complementary interactions between the mRNA stop codon and the grooves at different sides of the surface of the eRF1 fragment, containing helix α2, NIKS loop, and helix α3 of the N domain. In model 1, the nucleotides of the mRNA stop codon at the A site are approximately equidistant (～15 Å) from the N (motifs NIKS and YxCxxxF) and C domains. In model 2, the stop codon is close to the N-domain motifs NIKS and YxCxxxF. Both models fit genetic and biochemical experimental data. The choice of a particular model requires additional studies.     

Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation

By using single-molecule fluorescence resonance energy transfer (smFRET), we observe the real-time dynamic coupling between the ribosome, labeled at the L1 stalk, and transfer RNA (tRNA). We find that an interaction between the ribosomal L1 stalk and the newly deacylated tRNA is established spontaneously upon peptide bond formation; this event involves coupled movements of the L1 stalk and tRNAs as well as ratcheting of the ribosome. In the absence of elongation factor G, the entire pretranslocation ribosome fluctuates between just two states: a nonratcheted state, with tRNAs in their classical configuration and no L1 stalk-tRNA interaction, and a ratcheted state, with tRNAs in an intermediate hybrid configuration and a direct L1 stalk-tRNA interaction. We demonstrate that binding of EF-G shifts the equilibrium toward the ratcheted state. Real-time smFRET experiments reveal that the L1 stalk-tRNA interaction persists throughout the translocation reaction, suggesting that the L1 stalk acts to direct tRNA movements during translocation.     

Structural effects of modified ribonucleotides and magnesium in transfer RNAs

Modified nucleotides are ubiquitous and important to tRNA structure and function. To understand their effect on tRNA conformation, we performed a series of molecular dynamics simulations on yeast tRNA^Phe and tRNA_init, Escherichia coli tRNA_init and HIV tRNA^Lys. Simulations were performed with the wild type modified nucleotides, using the recently developed CHARMM compatible force field parameter set for modified nucleotides (J. Comput. Chem. 2016, 37, 896), or with the corresponding unmodified nucleotides, and in the presence or absence of Mg²⁺. Results showed a stabilizing effect associated with the presence of the modifications and Mg²⁺ for some important positions, such as modified guanosine in position 37 and dihydrouridines in 16/17 including both structural properties and base interactions. Some other modifications were also found to make subtle contributions to the structural properties of local domains. While we were not able to investigate the effect of adenosine 37 in tRNA_init and limitations were observed in the conformation of E. coli tRNA_init, the presence of the modified nucleotides and of Mg²⁺ better maintained the structural features and base interactions of the tRNA systems than in their absence indicating the utility of incorporating the modified nucleotides in simulations of tRNA and other RNAs.     

Experimental and computational determination of tRNA dynamics

As the molecular representation of the genetic code, tRNA plays a central role in the translational machinery where it interacts with several proteins and other RNAs during the course of protein synthesis. These interactions exploit the dynamic flexibility of tRNA. In this minireview, we discuss the effects of modified bases, ions, and proteins on tRNA structure and dynamics and the challenges of observing its motions over the cycle of translation.     

Structural insights into translational recoding by frameshift suppressor tRNASufJ

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5′ or 3′ direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA^SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA^SufJ contains an insertion 5′ to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL^SufJ or tRNA^SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL^SufJ and ASL^Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL^SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA^SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.     

Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.     

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNA^Asp and tRNA^Asn generating a correctly charged Asp-tRNA^Asp and an erroneous Asp-tRNA^Asn. This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNA^Asp over tRNA^Asn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNA^Asp and was unable to aspartylate tRNA^Asn. The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.     

A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA^Ala-based body (tRNA^AlaB) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA^PheB body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA^AlaB body than from the tRNA^PheB body. At ∼1 µM EF-Tu, tRNA^AlaB conferred considerably faster incorporation kinetics than tRNA^PheB, especially in the case of the bulky bK. In contrast, the swap to the tRNA^AlaB body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA^AlaB and tRNA^PheB bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.     

Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome

Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA^Asp analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3′-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.     

Motion of transfer RNA from the A/T state into the A‐site using docking and simulations

The ribosome catalyzes peptidyl transfer reactions at the growing nascent polypeptide chain. Here, we present a structural mechanism for selecting cognate over near‐cognate A/T transfer RNA (tRNA). In part, the structural basis for the fidelity of translation relies on accommodation to filter cognate from near‐cognate tRNAs. To examine the assembly of tRNAs within the ribonucleic–riboprotein complex, we conducted a series of all‐atom molecular dynamics (MD) simulations of the entire solvated 70S Escherichia coli ribosome, along with its associated cofactors, proteins, and messenger RNA (mRNA). We measured the motion of the A/T state of tRNA between initial binding and full accommodation. The mechanism of rejection was investigated. Using novel in‐house algorithms, we determined trajectory pathways. Despite the large intersubunit cavity, the available space is limited by the presence of the tRNA, which is equally large. This article describes a “structural gate,” formed between helices 71 and 92 on the ribosomal large subunit, which restricts tRNA motion. The gate and the interacting protein, L14, of the 50S ribosome act as steric filters in two consecutive substeps during accommodation, each requiring: (1) sufficient energy contained in the hybrid tRNA kink and (2) sufficient energy in the Watson–Crick base pairing of the codon–anticodon. We show that these barriers act to filter out near‐cognate tRNA and promote proofreading of the codon–anticodon. Since proofreading is essential for understanding the fidelity of translation, our model for the dynamics of this process has substantial biomedical implications. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Analysis of genomic tRNA revealed presence of novel genomic features in cyanobacterial tRNA

Transfer RNAs (tRNA) are important molecules that involved in protein translation machinery and acts as a bridge between the ribosome and codon of the mRNA. The study of tRNA is evolving considerably in the fields of bacteria, plants, and animals. However, detailed genomic study of the cyanobacterial tRNA is lacking. Therefore, we conducted a study of cyanobacterial tRNA from 61 species. Analysis revealed that; cyanobacteria contain thirty-six to seventy-eight tRNA gens per genome that encodes for 20 tRNA isotypes. The number of iso-acceptors (anti-codons) ranged from thirty-two to forty-three per genome. tRNA^Ile with anti-codon AAU, GAU, and UAU was reported to be absent from the genome of Gleocapsa PCC 73,106 and Xenococcus sp. PCC 7305. Instead, they were contained anti-codon CAU that is common to tRNA^Met and tRNA^Ile as well. The iso-acceptors ACA (tRNA^Cys), ACC (tRNA^Gly), AGA, ACU (tRNA^Ser), AAA (tRNA^Phe), AGG (tRNA^Pro), AAC (tRNA^Val), GCG (tRNA^Arg), AUG (tRNA^His), and AUC (tRNA^Asp) were absent from the genome of cyanobacterial lineages studied so far. A few of the cyanobacterial species encode suppressor tRNAs, whereas none of the species were found to encode a selenocysteine iso-acceptor. Cyanobacterial species encode a few putative novel tRNAs whose functions are yet to be elucidated.     

Ternary complex between elongation factor Tu•GTP and Phe-tRNA

The effect of aminoacylation and ternary complex formation with elongation factor Tu•GTP on the tertiary structure of yeast tRNA^Phe was examined by ¹H-NMR spectroscopy. Esterification of phenylalanine to tRNA^Phe does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA^Phe with elongation factor Tu•GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA^Phe upon binding to elongation factor Tu•GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA^Phe•elongation factor Tu•GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA^Phe preserves its typical L-shaped tertiary structure in the complex. At high (> 10⁻⁴ M) ligand concentrations a complex between tRNA^Phe and elongation factor Tu•GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA^Phe and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

Flipping of the Ribosomal A-Site Adenines Provides a Basis for tRNA Selection

Ribosomes control the missense error rate of ~ 10^− 4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near-cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3 kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4 kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA–tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA.