Barry Cooperman

• Ph.D.
• Professor (Full) at University of Pennsylvania

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in patient DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had...

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 mech...

Mitochondria maintain their own translational machinery that is responsible for the synthesis of essential components of the oxidative phosphorylation system. The mammalian mitochondrial translation system differs significantly from its cytosolic and bacterial counterparts. Here, we describe detailed protocols for efficient in vitro reconstitution...

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...

The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It has been commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation f...

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....

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 photoaffini...

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 photoaffini...

Significance Nonsense mutations giving rise to premature stop codons (PSCs) cause many diseases, creating the need to develop safe and effective translational read-through–inducing drugs (TRIDs). The current best-characterized TRIDs are ataluren and aminoglycosides. Only ataluren has been approved for clinical use, albeit in a limited context. Here...

Stress is known to induce retrograde tRNA translocation from the cytoplasm to the nucleus but translocation kinetics and tRNA-spatial distribution have not been characterized previously. We microinject fluorescently-labeled tRNA into living cells and use confocal microscopy to image tRNA spatial distribution in single cells at various levels of sta...

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...

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 comple...

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 th...

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 studie...

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...

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...

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...

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 interna...

Significance Elongation factor G (EF-G) uses energy stored in GTP to catalyze movement of transfer RNAs and messenger RNA in the ribosome during the translocation step of prokaryotic protein synthesis. Using single-molecule polarized fluorescence microscopy, three-dimensional rotational motions of individual domains of EF-G were directly captured,...

Initial coding sequences of variants used in this work. DOI: http://dx.doi.org/10.7554/eLife.13429.016

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 timescale with which it is deformed from equilibrium, as in the...

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)...

Intracellular RNA molecules adopt a wide variety of 3D structures, many of which have been studied using high-resolution structural techniques such as X-ray crystallography and NMR. However, while canonical structural features for a class of RNA molecules are assumed based on such studies, structural variations within a class are extremely difficul...

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 bein...

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...

During the polypeptide elongation cycle, prokaryotic elongation factor G (EF‐G) catalyzes the coupled translocations on the ribosome of mRNA and A‐ and P‐site bound tRNAs with high precision. However, the detailed dynamics of this important step have not been fully resolved. Here we describe the application of single molecule fluorescence resonance...

During the elongation cycle of protein synthesis, translocation of tRNAs and mRNA is catalyzed by the GTPase elongation factor G (EF-G) with high precision and speed. Conversion of the GTP to the GDP form of EF-G is considered essential for translocation, but the structural dynamics on the ribosome have not been reported. We used single molecule po...

Prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs during the polypeptide elongation cycle. However, the kinetic pathway of this important step has not been fully elucidated. Here we describe the application of single molecule fluorescence resonance energy transfer (smFR...

During protein synthesis, tRNAs successively occupy three sites on the ribosome: A (aminoacyl), P (peptidyl), and E (exit). A proposed model based on observations of stalled ribosomes describes two distinct states before mRNA and tRNA translocation (PRE-translocation): a classical state in which the tRNAs occupy the same sites in the large and smal...

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 transl...

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),...

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...

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...

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 very high signal-to-noise ratio. Such high r...

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 sp...

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 sp...

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 l...

Binding of tRNA to the E-site. (A) Non-specific E-site binding of Cy5.5-F in a buffer with polyamines (see Methods). Cy5.5-labeled deacylated Phe-tRNA was added to 70S initiation complex immobilized on the surface via biotin-labeled mRNA. Without EF-G, translation is halted, yet multiple binding events occur near the Cy3-labeled L1 protein. (B) A s...

Multiple criterion events detected by tRNA-tRNA FRET between Cy3-F and Cy5-V during translation of mRNA-3 (A), mRNA-4 (B), and mRNA-5 (C). Color coding as described in Fig. 1. (TIF)

Multiple criterion events detected by tRNA-tRNA FRET between Cy3-F and Cy5-V during translation of mRNA-6 (A). Analysis of mixtures of mRNA-2 and mRNA-6 (B). Color coding as described in Fig. 4. The criterion for classifying mRNA-6 from mRNA-2 is the occurrence of both FV and VF e (TIF)

Multiple criterion events detected by FRET between L1Cy3 and either Cy5.5-F or Cy5-V during translation of mRNA-3 (A), mRNA-4 (B), and mRNA-5 (C). Color coding as described in Fig. 2. (TIF)

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 substitut...

The rate of translation of full-length proteins by the ribosome influences expression levels, folding and frameshifting. In order to study the factors that control translation rates, we use the expression of fast maturing Emerald Green Fluorescent Protein (EmGFP) by a reconsitituted E. coli cell-free translation system. Single-molecule TIRF microsc...

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...

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....

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 localizatio...

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 la...

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 C...

The ribosome is a major target in the bacterial cell for antibiotics. Here, we dissect the effects that the thiopeptide antibiotics thiostrepton (ThS) and micro-coccin (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...

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...

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 fluctuatio...

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 t...

It has recently been suggested that peptidyl transferase activity is primarily a property of ribosomal RNA and that ribosomal proteins may act only as scaffolding. On the other hand, evidence from both photoaffinity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the wor...

Continued dramatic progress in the elucidation of the structures of the bacterial ribosome and its functional complexes has led to proposals for the detailed mechanisms of ribosome-catalyzed protein synthesis (Schmeing and Ramakrishnan, 2009; Agirrezabala and Frank, 2009). Ensemble rapid reaction kinetics (Antoun et al., 2006; Daviter et al., 2006;...

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 ha...

We employ single‐molecule TIRF spectroscopy to study ribosomal structural dynamics over the first two elongation cycles, using complexes containing either Cy3‐labeled L11 and Cy5‐labeled tRNAs to monitor dynamics between L11 and P‐ or A‐site tRNAs (Lt complexes) or Cy3 and Cy5 labeled tRNAs to monitor dynamics between adjacent tRNAs (tt complexes)....

The QSY9 derivative of E348C‐EF‐Tu (EF‐Tu Q348 ) is fully functional and is an efficient quencher of Cy3 fluorescence within the ternary complex made with Phe‐tRNA Phe (Cy3). Incubation of this ternary complex with a ribosomal initiation complex leads to a large increase in Cy3 fluorescence as EF‐Tu Q348 is released from the ribosome and Phe‐tRNA P...

EF4 (LepA) 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‐catal...

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,...

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...

During ribosome-catalyzed polypeptide chain elongation, dissociation of the deacylated tRNA from the E-site has been proposed to be either spontaneous or triggered allosterically by binding of the next cognate ternary complex to the A-site. Using fluorescent labeled tRNAs, we have measured single molecule fluorescence intensities and single molecul...

Metal enhanced fluorescence (MEF), in which a surface plasmon near a noble metal alters the spectral properties of an organic fluorophore, increases fluorescence intensity without a concomitant increase in photobleaching rate. To improve recordings of single molecule fluorescence signals from individual ribosomes, we studied the emission of Cy3- an...

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 thio...

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 forma-tion. We have used several in vitro translational machinery assays to screen a library of thi...

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 c...

We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthes...

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The ribosome is the macromolecular machine responsible for translating the genetic code into polypeptide chains. Despite impressive structural and kinetic studies of the translation process, a number of challenges remain with respect to understanding the dynamic properties of the translation apparatus. Single-molecule techniques hold the potential...

All mature tRNA molecules have the conserved CCA sequence at the 3' end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dyn...

Eukaryotic ribonucleotide reductase (RR) catalyzes nucleoside diphosphate conversion to deoxynucleoside diphosphate. Crucial for rapidly dividing cells, RR is a target for cancer therapy. RR activity requires formation of a complex between subunits R1 and R2 in which the R2 C-terminal peptide binds to R1. Here we report crystal structures of hetero...

The papain/CLIK-148 coordinate system was employed as a model to study the interactions of a nonpeptide thiocarbazate inhibitor of cathepsin L ( 1). This small molecule inhibitor, a thiol ester containing a diacyl hydrazine functionality and one stereogenic center, was most active as the S-enantiomer, with an IC 50 of 56 nM; the R-enantiomer ( 2) d...

During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at posi...

A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.15) with an IC(50) of 56 nM, was developed after a 57,821-compound screen of the National Institutes of Health Molecular Libraries Small Molecule Repository. After a 4-h preincubation with cathepsin L, this compound became even more poten...

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-tRNAPhe, 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of amin...

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small...

Metallic particles, silver in particular, can significantly enhance the fluorescence of dye molecules in the immediate vicinity (5-20 nm) of the particle. This magnifying effect can be theoretically explained/predicted by considering the change of photonic mode density near the fluorophore due to coupling to the conducting surface. We are using thi...

Association of the 30 S initiation complex (30SIC) and the 50 S ribosomal subunit, leading to formation of the 70 S initiation complex (70SIC), is a critical step of the translation initiation pathway. The 70SIC contains initiator tRNA, fMet-tRNA(fMet), bound in the P (peptidyl)-site in response to the AUG start codon. We have formulated a quantita...