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Background: A common tibial construct for revision total knee arthroplasty includes a long diaphyseal engaging press-fit stem. Due to tibial canal bowing, compromises are often necessary to match patient anatomy when choosing stemmed implants. The objective of this study is to determine through 3-D modeling whether current implant press-fit options appropriately fit patient anatomy, or whether an alternative angle between the stem and baseplate could increase the cortical engagement of long press-fit tibial stems. Methods: Preoperative computerized tomography scans from 100 patients undergoing TKA were imported into an image-processing software program. Three-dimensional models were created with tibial stems placed at a fixed perpendicular angle and a custom angle to the revision tibial baseplate. Stem diameter, depth, offset, and contact surface area were measured and analyzed between the 2 groups. Results: Significantly more cortical contact, larger stem diameter, and smaller offset of the custom keel from the center of the baseplate were associated with free custom tibial stem placement vs a fixed perpendicular baseplate-stem interface (P < .001). Statistically significant differences were also found between different patient demographics. Conclusions: Custom free-angle stem placement allows for increased stem diameter and cortical contact of press-fit tibial stems compared to existing constructs that must interface with the baseplate at a 90-degree angle. Current revision tibia implants limit fixation of tibial press-fit stems and often mismatch with patient anatomy. Alternative ways to fit patient anatomy may be beneficial for patients with extreme mismatch. In the future, custom keel angles may help to resolve this problem.

The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with an in vitro eukaryotic translation system to investigate tRNA selection and subsequent steps during peptide elongation. We compared accommodation of a tryptophan-aminoacyl-tRNA into the ribosomal A-site containing either a cognate or near-cognate codon and unexpectedly found that, following an initial slow sampling event, subsequent near-cognate sampling events proceeded more rapidly than the initial event. Further, we found a strong negative correlation between the concentration of near-cognate aminoacyl-tRNA and the efficiency of tRNA accommodation. These novel characteristics of near-cognate interaction with the eukaryotic ribosome suggest that rejection of a near-cognate tRNAs leads to formation of an altered ribosomal conformation that assists in rejecting subsequent incorrect tRNA interactions., Competing Interests: Conflict of interest statement. None declared.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein, we identify the molecular mechanisms involved, showing that TRAP1 (1) binds both mitochondrial and cytosolic ribosomes, as well as translation elongation factors; (2) slows down translation elongation rate; and (3) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration., (© 2023 Avolio et al.; Published by Cold Spring Harbor Laboratory Press.)

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren's mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren's binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren's low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria., (© 2023. The Author(s).)

Genetic diseases are often caused by nonsense mutations, but only one TRID (translation readthrough inducing drug), ataluren, has been approved for clinical use. Ataluren inhibits release factor complex (RFC) termination activity, while not affecting productive binding of near-cognate ternary complex (TC, aa-tRNA.eEF1A.GTP). Here we use photoaffinity labeling to identify two sites of ataluren binding within rRNA, proximal to the decoding center (DC) and the peptidyl transfer center (PTC) of the ribosome, which are directly responsible for ataluren inhibition of termination activity. A third site, within the RFC, has as yet unclear functional consequences. Using single molecule and ensemble fluorescence assays we also demonstrate that termination proceeds via rapid RFC-dependent hydrolysis of peptidyl-tRNA followed by slow release of peptide and tRNA from the ribosome. Ataluren is an apparent competitive inhibitor of productive RFC binding, acting at or before the hydrolysis step. We propose that designing more potent TRIDs which retain ataluren's low toxicity should target areas of the RFC binding site proximal to the DC and PTC which do not overlap the TC binding site., (© 2022. The Author(s).)

The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs. In addition, several methods are described in which specifically labeled RNAs are prepared de novo.

During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers. Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously [1], we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell's protein synthesis machinery. Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near-cognate tRNA ± TRIDs., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Nonsense suppressors (NonSups) induce "readthrough", i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy., Competing Interests: The authors declare no competing financial interest.

Downstream stable mRNA secondary structures can stall elongating ribosomes by impeding the concerted movements of tRNAs and mRNA on the ribosome during translocation. The addition of a downstream mRNA structure, such as a stem-loop or a pseudoknot, is essential to induce -1 programmed ribosomal frameshifting (-1 PRF). Interestingly, previous studies revealed that -1 PRF efficiencies correlate with conformational plasticity of pseudoknots, defined as their propensity to form incompletely folded structures, rather than with the mechanical properties of pseudoknots. To elucidate the detailed molecular mechanisms of translocation and -1 PRF, we applied several smFRET assays to systematically examine how translocation rates and conformational dynamics of ribosomes were affected by different pseudoknots. Our results show that initial pseudoknot-unwinding significantly inhibits late-stage translocation and modulates conformational dynamics of ribosomal post-translocation complexes. The effects of pseudoknots on the structural dynamics of ribosomes strongly correlate with their abilities to induce -1 PRF. Our results lead us to propose a kinetic scheme for translocation which includes an initial power-stroke step and a following thermal-ratcheting step. This scheme provides mechanistic insights on how selective modulation of late-stage translocation by pseudoknots affects -1 PRF. Overall our findings advance current understanding of translocation and ribosome-induced mRNA structure unwinding.

According to the traditional view, GTPases act as molecular switches, which cycle between distinct 'on' and 'off' conformations bound to GTP and GDP, respectively. Translation elongation factor EF-Tu is a GTPase essential for prokaryotic protein synthesis. In its GTP-bound form, EF-Tu delivers aminoacylated tRNAs to the ribosome as a ternary complex. GTP hydrolysis is thought to cause the release of EF-Tu from aminoacyl-tRNA and the ribosome due to a dramatic conformational change following Pi release. Here, the crystal structure of Escherichia coli EF-Tu in complex with a non-hydrolysable GTP analogue (GDPNP) has been determined. Remarkably, the overall conformation of EF-Tu·GDPNP displays the classical, open GDP-bound conformation. This is in accordance with an emerging view that the identity of the bound guanine nucleotide is not 'locking' the GTPase in a fixed conformation. Using a single-molecule approach, the conformational dynamics of various ligand-bound forms of EF-Tu were probed in solution by fluorescence resonance energy transfer. The results suggest that EF-Tu, free in solution, may sample a wider set of conformations than the structurally well-defined GTP- and GDP-forms known from previous X-ray crystallographic studies. Only upon binding, as a ternary complex, to the mRNA-programmed ribosome, is the well-known, closed GTP-bound conformation, observed.

The GTPase elongation factor EF-Tu delivers aminoacyl-tRNAs to the mRNA-programmed ribosome during translation. Cognate codon-anticodon interaction stimulates GTP hydrolysis within EF-Tu. It has been proposed that EF-Tu undergoes a large conformational change subsequent to GTP hydrolysis, which results in the accommodation of aminoacyl-tRNA into the ribosomal A-site. However, this proposal has never been tested directly. Here, we apply single-molecule total internal reflection fluorescence microscopy to study the conformational dynamics of EF-Tu when bound to the ribosome. Our studies show that GTP hydrolysis initiates a partial, comparatively small conformational change of EF-Tu on the ribosome, not directly along the path from the solution 'GTP' to the 'GDP' structure. The final motion is completed either concomitant with or following dissociation of EF-Tu from the ribosome. The structural transition of EF-Tu on the ribosome is slower when aa-tRNA binds to a cognate versus a near-cognate codon. The resulting longer residence time of EF-Tu on the ribosome may be important for promoting accommodation of the cognate aminoacyl-tRNA into the A-site.

The pretranslocation complex of the ribosome can undergo spontaneous fluctuations of messenger RNA and transfer RNAs (tRNAs) between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classical and hybrid state tRNA positions have been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation. However, during multiple ongoing elongation cycles when both EF-G and ternary complexes are present, EF-G can bind to the pretranslocation complex much faster than the timescale of the classic-hybrid transitions. Using single-molecule fluorescence resonance energy transfer between adjacent tRNAs and between A-site tRNA and ribosomal protein L11, we found that the tRNAs do not fluctuate between the hybrid and classical states, but instead adopt a position with fluorescence resonance energy transfer efficiencies between those of the stalled classical and hybrid states., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs. Our results largely reconcile these previously held views. We demonstrate that, in the absence of an upstream Shine-Dalgarno (SD) sequence, PoTC breakdown proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting. By contrast, in the presence of an SD sequence all three processes proceed with identical apparent rates, with the splitting step likely being rate-determining. Our results are consistent with ribosome profiling results demonstrating the influence of upstream SD-like sequences on ribosome occupancy at or just before the mRNA stop codon., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Accurate translation of the genetic code depends on mRNA:tRNA codon:anticodon base pairing. Here we exploit an emissive, isosteric adenosine surrogate that allows direct measurement of the kinetics of codon:anticodon University of California base formation during protein synthesis. Our results suggest that codon:anticodon base pairing is subject to tighter constraints at the middle position than at the 5'- and 3'-positions, and further suggest a sequential mechanism of formation of the three base pairs in the codon:anticodon helix., Competing Interests: The authors declare no competing financial interests.

Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm 2 /s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G's GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

The intergenic IRES of Cricket Paralysis Virus (CrPV-IRES) forms a tight complex with 80S ribosomes capable of initiating the cell-free synthesis of complete proteins in the absence of initiation factors. Such synthesis raises the question of what effect the necessary IRES dissociation from the tRNA binding sites, and ultimately from all of the ribosome, has on the rates of initial peptide elongation steps as nascent peptide is formed. Here we report the first results measuring rates of reaction for the initial cycles of IRES-dependent elongation. Our results demonstrate that 1) the first two cycles of elongation proceed much more slowly than subsequent cycles, 2) these reduced rates arise from slow pseudo-translocation and translocation steps, and 3) the retarding effect of ribosome-bound IRES on protein synthesis is largely overcome following translocation of tripeptidyl-tRNA. Our results also provide a straightforward approach to detailed mechanistic characterization of many aspects of eukaryotic polypeptide elongation.

Over half of all antibiotics target the bacterial ribosome-nature's complex, 2.5 MDa nanomachine responsible for decoding mRNA and synthesizing proteins. Macrolide antibiotics, exemplified by erythromycin, bind the 50S subunit with nM affinity and inhibit protein synthesis by blocking the passage of nascent oligopeptides. Solithromycin (1), a third-generation semisynthetic macrolide discovered by combinatorial copper-catalyzed click chemistry, was synthesized in situ by incubating either E. coli 70S ribosomes or 50S subunits with macrolide-functionalized azide 2 and 3-ethynylaniline (3) precursors. The ribosome-templated in situ click method was expanded from a binary reaction (i.e., one azide and one alkyne) to a six-component reaction (i.e., azide 2 and five alkynes) and ultimately to a 16-component reaction (i.e., azide 2 and 15 alkynes). The extent of triazole formation correlated with ribosome affinity for the anti (1,4)-regioisomers as revealed by measured Kd values. Computational analysis using the site-identification by ligand competitive saturation (SILCS) approach indicated that the relative affinity of the ligands was associated with the alteration of macrolactone+desosamine-ribosome interactions caused by the different alkynes. Protein synthesis inhibition experiments confirmed the mechanism of action. Evaluation of the minimal inhibitory concentrations (MIC) quantified the potency of the in situ click products and demonstrated the efficacy of this method in the triaging and prioritization of potent antibiotics that target the bacterial ribosome. Cell viability assays in human fibroblasts confirmed 2 and four analogues with therapeutic indices for bactericidal activity over in vitro mammalian cytotoxicity as essentially identical to solithromycin (1).

It has been hypothesized that the ribosome gains additional fidelity during protein translation by probing structural differences in tRNA species. We measure the translocation kinetics of different tRNA species through ∼3 nm diameter synthetic nanopores. Each tRNA species varies in the time scale with which it is deformed from equilibrium, as in the translocation step of protein translation. Using machine-learning algorithms, we can differentiate among five tRNA species, analyze the ratios of tRNA binary mixtures, and distinguish tRNA isoacceptors.

Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs.

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 Pi 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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells., (© 2014 Wiley Periodicals, Inc.)

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching to tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA(Ile)UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.

Here we examine three mRNAs, site-specifically modified at codon positions 4, 5, and 6 with a new emissive and responsive isosteric guanosine mimetic ((th)G), with the goal of developing real time assays for monitoring translation-related events at nucleotide resolution. All three emissive mRNAs tested form initiation (70SIC), pretranslocation (PRE), and posttranslocation (POST) complexes. In most cases spectral differences are seen on binding of the mRNA to the ribosome during 70SIC formation and on conversion of 70SIC to PRE complexes and PRE complexes to POST complexes. These differences allow measurement of the kinetics of such conversions by changes in the fluorescence of labeled mRNAs. Such measurements directly identify a specific step during PRE complex formation, provisionally assigned to codon:anticodon-loop base pair formation, that follows aa-tRNA.EF-Tu.GTP ternary complex binding to the ribosome and precedes aa-tRNA accommodation into the A-site of the ribosome. These observations demonstrate not only the functionality of mRNAs modified with the emissive guanosine mimetic but also the potential this mimetic offers for observing the formation and disappearance of discrete intermediates during the polypeptide elongation cycle.

Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification.

When suitably labeled bulk tRNAs are transfected into cells they give rise to FRET (fluorescence resonance energy transfer) signals via binding to ribosomes that provide a measure of total protein synthesis. Application of this approach to monitoring rates of specific protein synthesis requires achieving a very high signal-to-noise ratio. Such high ratios may be attainable using LRET (luminescence resonance energy transfer) in place of FRET. Lanthanide complexes containing an antenna chromophore are excellent LRET donors. Here we describe the synthesis of a Phe-tRNA(Phe) labeled with a Tb(3+) complex, denoted Tb(3+)-Phe-tRNA(Phe) that, notwithstanding the bulkiness of the Tb(3+) complex, is active in protein synthesis.

During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

We present a flexible, real-time-coupled transcription-translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.

We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically released when the next aminoacyl tRNA binds to the A site. This process is regulated by the length and sequence of the nascent peptide and by the conformational state, detected by tRNA proximity, prior to translocation. In later cycles, E-site tRNA dissociates spontaneously. Our results suggest that the distribution of pretranslocation tRNA states and posttranslocation pathways are correlated within each elongation cycle via communication between distant subdomains in the ribosome, but that this correlation between elongation cycle intermediates does not persist into succeeding cycles.

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

EF4 (LepA), a strongly conserved protein, is important for bacterial growth and functional protein biosynthesis under certain conditions and is quite similar structurally to the translocase EF-G. The elongation cycle in protein synthesis is characterized by ribosome oscillation between pretranslocation (PRE) and posttranslocation (POST) complexes. Here, using ensemble single turnover and equilibrium experiments, as well as single molecule FRET measurements, we demonstrate that EF4 can compete with EF-G for binding to the PRE complex. Such EF4 binding results in formation of a complex, denoted X(3), that effectively sequesters a catalytically active ribosome, leading to a transient inhibition of elongation that provides a mechanism for optimization of functional protein synthesis. Earlier [Liu H, et al. (2010) J Mol Biol 396:1043-1052] we demonstrated that EF4 also reacts with POST complex, leading to the formation of a complex, I(3), that appears to be identical with X(3). Our present results strongly suggest that PRE complex is the principal target of EF4 action on translation, rather than POST complex as had been previously supposed.

Dihydrouridine (DHU) positions within tRNAs have long been used as sites to covalently attach fluorophores, by virtue of their unique chemical reactivity toward reduction by NaBH(4), their abundance within prokaryotic and eukaryotic tRNAs, and the biochemical functionality of the labeled tRNAs so produced. Interpretation of experiments employing labeled tRNAs can depend on knowing the distribution of dye among the DHU positions present in a labeled tRNA. Here we combine matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis of oligonucleotide fragments and thin layer chromatography to resolve and quantify sites of DHU labeling by the fluorophores Cy3, Cy5, and proflavin in Escherichia coli tRNA(Phe) and E. coli tRNA(Arg). The MALDI-MS results led us to re-examine the precise chemistry of the reactions that result in fluorophore introduction into tRNA. We demonstrate that, in contrast to an earlier suggestion that has long been unchallenged in the literature, such introduction proceeds via a substitution reaction on tetrahydrouridine, the product of NaBH(4) reduction of DHU, resulting in formation of substituted tetrahydrocytidines within tRNA.

The ribosome is a major target in the bacterial cell for antibiotics. Here, we dissect the effects that the thiopeptide antibiotics thiostrepton (ThS) and micrococcin (MiC) as well as the orthosomycin antibiotic evernimicin (Evn) have on translational GTPases. We demonstrate that, like ThS, MiC is a translocation inhibitor, and that the activation by MiC of the ribosome-dependent GTPase activity of EF-G is dependent on the presence of the ribosomal proteins L7/L12 as well as the G' subdomain of EF-G. In contrast, Evn does not inhibit translocation but is a potent inhibitor of back-translocation as well as IF2-dependent 70S-initiation complex formation. Collectively, these results shed insight not only into fundamental aspects of translation but also into the unappreciated specificities of these classes of translational inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Metal-enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold, respectively. Fluorescence intensity fluctuations above shot noise, at 0.1-5 Hz, were greater on silver particles. Overall signal-to-noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G.

Although the nucleotides in tRNA required for aminoacylation are conserved in evolution, bacterial aminoacyl-transfer RNA synthetases are unable to acylate eukaryotic tRNA. The cross-species barrier may be due to the absence of eukaryote-specific domains from bacterial aminoacyl-transfer RNA synthetases. Here we show that whereas Escherichia coli CysRS cannot acylate human tRNA(Cys), the fusion of a eukaryote-specific domain of human CysRS overcomes the cross-species barrier in human tRNA(Cys). In addition to enabling recognition of the sequence differences in the tertiary core of tRNA(Cys), the fused eukaryotic domain redirects the specificity of E. coli CysRS from the A37 present in bacterial tRNA(Cys) to the G37 in mammals. Further experiments show that the accuracy of codon recognition on the ribosome was also highly sensitive to the A37G transition in tRNA(Cys). These results raise the possibility of the development of tRNA nucleotide determinants for aminoacylation being interdependent with those for ribosome decoding.

The translational apparatus of the bacterial cell remains one of the principal targets of antibiotics for the clinical treatment of infection worldwide. Since the introduction of specific translation inhibitors into clinical practice in the late 1940s, intense efforts have been made to understand their precise mechanisms of action. Such research has often revealed significant and sometimes unexpected insights into many fundamental aspects of the translation mechanism. Central to progress in this area, high-resolution crystal structures of the bacterial ribosome identifying the sites of antibiotic binding are now available, which, together with recent developments in single-molecule and fast-kinetic approaches, provide an integrated view of the dynamic translation process. Assays employing these approaches and focusing on specific steps of the overall translation process are amenable for drug screening. Such assays, coupled with structural studies, have the potential not only to accelerate the discovery of novel and effective antimicrobial agents, but also to refine our understanding of the mechanisms of translation. Antibiotics often stabilize specific functional states of the ribosome and therefore allow distinct translation steps to be dissected in molecular detail.

EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST-->I(1)-->I(2)-->I(3)-->PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3'-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I(3) rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I(3) is likely to be important for optimizing functional protein biosynthesis., ((c) 2010 Elsevier Ltd. All rights reserved.)

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

Most thiopeptide antibiotics target the translational machinery: thiostrepton (ThS) and nosiheptide (NoS) target the ribosome and inhibit translation factor function, whereas GE2270A/T binds to the elongation factor EF-Tu and prevents ternary complex formation. We have used several in vitro translational machinery assays to screen a library of thiopeptide antibiotic precursor compounds and identified four families of precursor compounds that are either themselves inhibitory or are able to relieve the inhibitory effects of ThS, NoS, or GE2270T. Some of these precursors represent distinct compounds with respect to their ability to bind to ribosomes. The results not only provide insight into the mechanism of action of thiopeptide compounds but also demonstrate the potential of such assays for identifying lead compounds that might be missed using conventional inhibitory screening protocols.

Addition of an Escherichia coli 50S subunit (50S(Cy5)) containing a Cy5-labeled L11 N-terminal domain (L11-NTD) within the GTPase-associated center (GAC) to an E. coli 30S initiation complex (30SIC(Cy3)) containing Cy3-labeled initiation factor 2 complexed with GTP leads to rapid development of a FRET signal during formation of the 70S initiation complex (70SIC). Initiation factor 2 (IF2) and elongation factor G (EF-G) induce similar changes in ribosome structure. Here we show that such similarities are maintained on a dynamic level as well. Thus, movement of IF2 toward L11-NTD after initial 70S ribosome formation follows GTP hydrolysis and precedes P(i) release, paralleling movement of EF-G following its binding to the ribosome [Seo, H., et al. (2006) Biochemistry 45, 2504-2514], and in both cases, the rate of such movement is slowed if GTP hydrolysis is prevented. The 30SIC(Cy3):50S(Cy5) FRET signal also provides a sensitive probe of the ability of initiation factor 3 to discriminate between a canonical and a noncanonical initiation codon during 70SIC formation. We employ Bacillus stearothermophilus IF2 as a substitute for E. coli IF2 to take advantage of the higher stability of the complexes it forms with E. coli ribosomes. While Bst-IF2 is fully functional in formation of E. coli 70SIC, relative reactivities toward dipeptide formation of 70SICs formed with the two IF2s suggest that the Bst-IF2.GDP complex is more difficult to displace from the GAC than the E. coli IF2.GDP complex.