Education & Certifications

  • Ph.D., Cornell University, Chemistry and Chemical Biology (2010)
  • MS, Cornell University, Chemistry and Chemical Biology (2008)
  • B.Sc.(Hons), University College Dublin, Ireland, Chemistry (2005)


All Publications

  • Multiple Parallel Pathways of Translation Initiation on the CrPV IRES. Molecular cell Petrov, A., Grosely, R., Chen, J., O'Leary, S. E., Puglisi, J. D. 2016; 62 (1): 92-103


    The complexity of eukaryotic translation allows fine-tuned regulation of protein synthesis. Viruses use internal ribosome entry sites (IRESs) to minimize or, like the CrPV IRES, eliminate the need for initiation factors. Here, by exploiting the CrPV IRES, we observed the entire process of initiation and transition to elongation in real time. We directly tracked the CrPV IRES, 40S and 60S ribosomal subunits, and tRNA using single-molecule fluorescence spectroscopy and identified multiple parallel initiation pathways within the system. Our results distinguished two pathways of 80S:CrPV IRES complex assembly that produce elongation-competent complexes. Following 80S assembly, the requisite eEF2-mediated translocation results in an unstable intermediate that is captured by binding of the elongator tRNA. Whereas initiation can occur in the 0 and +1 frames, the arrival of the first tRNA defines the reading frame and strongly favors 0 frame initiation. Overall, even in the simplest system, an intricate reaction network regulates translation initiation.

    View details for DOI 10.1016/j.molcel.2016.03.020

    View details for PubMedID 27058789

  • N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics. Nature structural & molecular biology Choi, J., Ieong, K., Demirci, H., Chen, J., Petrov, A., Prabhakar, A., O'Leary, S. E., Dominissini, D., Rechavi, G., Soltis, S. M., Ehrenberg, M., Puglisi, J. D. 2016; 23 (2): 110-115


    N(6)-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

    View details for DOI 10.1038/nsmb.3148

    View details for PubMedID 26751643

  • Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions CELL Chen, J., Coakley, A., O'Connor, M., Petrov, A., O'Leary, S. E., Atkins, J. F., Puglisi, J. D. 2015; 163 (5): 1267-1280
  • Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fuchs, G., Petrov, A. N., Marceau, C. D., Popov, L. M., Chen, J., O'Leary, S. E., Wang, R., Carette, J. E., Sarnow, P., Puglisi, J. D. 2015; 112 (2): 319-325


    Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

    View details for DOI 10.1073/pnas.1421328111

    View details for Web of Science ID 000347732300029

    View details for PubMedID 25516984

  • Dynamic pathways of -1 translational frameshifting. Nature Chen, J., Petrov, A., Johansson, M., Tsai, A., O'Leary, S. E., Puglisi, J. D. 2014; 512 (7514): 328-332


    Spontaneous changes in the reading frame of translation are rare (frequency of 10(-3) to 10(-4) per codon), but can be induced by specific features in the messenger RNA (mRNA). In the presence of mRNA secondary structures, a heptanucleotide 'slippery sequence' usually defined by the motif X XXY YYZ, and (in some prokaryotic cases) mRNA sequences that base pair with the 3' end of the 16S ribosomal rRNA (internal Shine-Dalgarno sequences), there is an increased probability that a specific programmed change of frame occurs, wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame (-1 frame) and continues by translating a new sequence of amino acids. Despite extensive biochemical and genetic studies, there is no clear mechanistic description for frameshifting. Here we apply single-molecule fluorescence to track the compositional and conformational dynamics of individual ribosomes at each codon during translation of a frameshift-inducing mRNA from the dnaX gene in Escherichia coli. Ribosomes that frameshift into the -1 frame are characterized by a tenfold longer pause in elongation compared to non-frameshifted ribosomes, which translate through unperturbed. During the pause, interactions of the ribosome with the mRNA stimulatory elements uncouple EF-G catalysed translocation from normal ribosomal subunit reverse-rotation, leaving the ribosome in a non-canonical intersubunit rotated state with an exposed codon in the aminoacyl-tRNA site (A site). tRNA(Lys) sampling and accommodation to the empty A site and EF-G action either leads to the slippage of the tRNAs into the -1 frame or maintains the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for -1 frameshifting, highlighting multiple kinetic branchpoints during elongation.

    View details for DOI 10.1038/nature13428

    View details for PubMedID 24919156

  • Dynamic pathways of-1 translational frameshifting NATURE Chen, J., Petrov, A., Johansson, M., Tsai, A., O'Leary, S. E., Puglisi, J. D. 2014; 512 (7514): 328-?
  • High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, J., Dalal, R. V., Petrov, A. N., Tsai, A., O'Leary, S. E., Chapin, K., Cheng, J., Ewan, M., Hsiung, P., Lundquist, P., Turner, S. W., Hsu, D. R., Puglisi, J. D. 2014; 111 (2): 664-669


    Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

    View details for DOI 10.1073/pnas.1315735111

    View details for Web of Science ID 000329614500033

  • Dynamic Recognition of the mRNA Cap by Saccharomyces cerevisiae eIF4E STRUCTURE O'Leary, S. E., Petrov, A., Chen, J., Puglisi, J. D. 2013; 21 (12): 2197-2207


    Recognition of the mRNA 5' m⁷G(5')ppp(5')N cap is key to translation initiation for most eukaryotic mRNAs. The cap is bound by the eIF4F complex, consisting of a cap-binding protein (eIF4E), a "scaffold" protein (eIF4G), and an RNA helicase (eIF4A). As a central early step in initiation, regulation of eIF4F is crucial for cellular viability. Although the structure and function of eIF4E have been defined, a dynamic mechanistic picture of its activity at the molecular level in the eIF4F·mRNA complex is still unavailable. Here, using single-molecule fluorescence, we measured the effects of Saccharomyces cerevisiae eIF4F factors, mRNA secondary structure, and the poly(A)-binding protein Pab1p on eIF4E-mRNA binding dynamics. Our data provide an integrated picture of how eIF4G and mRNA structure modulate eIF4E-mRNA interaction, and uncover an eIF4G- and poly(A)-independent activity of poly(A)-binding protein that prolongs the eIF4E·mRNA complex lifetime.

    View details for DOI 10.1016/j.str.2013.09.016

    View details for Web of Science ID 000328914900013

  • Disassembly of All SNARE Complexes by N-Ethylmaleimide-sensitive Factor (NSF) Is Initiated by a Conserved 1:1 Interaction between alpha-Soluble NSF Attachment Protein (SNAP) and SNARE Complex JOURNAL OF BIOLOGICAL CHEMISTRY Vivona, S., Cipriano, D. J., O'Leary, S., Li, Y. H., Fenn, T. D., Brunger, A. T. 2013; 288 (34): 24984-24991


    Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF and the adapter protein α-SNAP disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complex and one binary complex we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and ternary SNARE complex form a 1:1 complex as revealed by multi-angle light scattering. We propose a model of NSF-mediated disassembly, where the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

    View details for DOI 10.1074/jbc.M113.489807

    View details for Web of Science ID 000330612300061

    View details for PubMedID 23836889

  • Coordinated conformational and compositional dynamics drive ribosome translocation. Nature structural & molecular biology Chen, J., Petrov, A., Tsai, A., O'Leary, S. E., Puglisi, J. D. 2013; 20 (6): 718-727


    During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G bound to GTP (EF-G-GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G-ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G-GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.

    View details for DOI 10.1038/nsmb.2567

    View details for PubMedID 23624862

  • Coordinated conformational and compositional dynamics drive ribosome translocation NATURE STRUCTURAL & MOLECULAR BIOLOGY Chen, J., Petrov, A., Tsai, A., O'Leary, S. E., Puglisi, J. D. 2013; 20 (6): 718-?

    View details for DOI 10.1038/nsmb.2567

    View details for Web of Science ID 000319915900013

  • Structural and Mechanistic Studies of HpxO, a Novel Flavin Adenine Dinucleotide-Dependent Urate Oxidase from Klebsiella pneumoniae BIOCHEMISTRY Hicks, K. A., O'Leary, S. E., Begley, T. P., Ealick, S. E. 2013; 52 (3): 477-487


    HpxO is a flavin-dependent urate oxidase that catalyzes the hydroxylation of uric acid to 5-hydroxyisourate and functions in a novel pathway for purine catabolism found in Klebsiella pneumoniae. We have determined the structures of HpxO with and without uric acid at 2.0 and 2.2 Å, respectively. We have also determined the structure of the R204Q variant at 2.0 Å resolution in the absence of uric acid. The variant structure is very similar to that of wild-type HpxO except for the conformation of Arg103, which interacts with FAD in the variant but not in the wild-type structure. Interestingly, the R204Q variant results in the uncoupling of nicotinamide adenine dinucleotide oxidation from uric acid hydroxylation. This suggests that Arg204 facilitates the deprotonation of uric acid, activating it for the oxygen transfer. On the basis of these data, a mechanism for this reaction consisting of a nucleophilic attack of the urate anion on the flavin hydroperoxide resulting in the formation of 5-hydroxyisourate is proposed.

    View details for DOI 10.1021/bi301262p

    View details for Web of Science ID 000314082700004

    View details for PubMedID 23259842

  • Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones. The Journal of biological chemistry Gokulan, K., O'Leary, S. E., Russell, W. K., Russell, D. H., Lalgondar, M., Begley, T. P., Ioerger, T. R., Sacchettini, J. C. 2013


    PKS11 is one of three type III polyketide synthases identified in M. tuberculosis (Mtb). Although many PKSs in Mtb have been implicated in producing complex cell-wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty-acid substrates. We solved the crystal structure of Mtb PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys138 to accommodate substrates. We observed electron density in this tunnel from a copurified molecule that was identified by mass spectrometry to be palmitate. Cocrystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methylbranched alkylpyrones.

    View details for PubMedID 23615910

  • Unraveling the dynamics of ribosome translocation CURRENT OPINION IN STRUCTURAL BIOLOGY Chen, J., Tsai, A., O'Leary, S. E., Petrov, A., Puglisi, J. D. 2012; 22 (6): 804-814


    Translocation is one of the key events in translation, requiring large-scale conformational changes in the ribosome, movements of two transfer RNAs (tRNAs) across a distance of more than 20?, and the coupled movement of the messenger RNA (mRNA) by one codon, completing one cycle of peptide-chain elongation. Translocation is catalyzed by elongation factor G (EF-G in bacteria), which hydrolyzes GTP in the process. However, how the conformational rearrangements of the ribosome actually drive the movements of the tRNAs and how EF-G GTP hydrolysis plays a role in this process are still unclear. Fluorescence methods, both single-molecule and bulk, have provided a dynamic view of translocation, allowing us to follow the different conformational changes of the ribosome in real-time. The application of electron microscopy has revealed new conformational intermediates during translocation and important structural rearrangements in the ribosome that drive tRNA movement, while computational approaches have added quantitative views of the translational pathway. These recent advances shed light on the process of translocation, providing insight on how to resolve the different descriptions of translocation in the current literature.

    View details for DOI 10.1016/

    View details for Web of Science ID 000312421000015

    View details for PubMedID 23142574

  • Single-Molecule Analysis of Translational Dynamics COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Petrov, A., Chen, J., O'Leary, S., Tsai, A., Puglisi, J. D. 2012; 4 (9)


    Decades of extensive biochemical and biophysical research have outlined the mechanism of translation. Rich structural studies have provided detailed snapshots of the translational machinery at all phases of the translation cycle. However, the relationship between structural dynamics, composition, and function remains unknown. The multistep nature of each stage of the translation cycle results in rapid desynchronization of individual ribosomes, thus hindering elucidation of the underlying mechanisms by conventional bulk biophysical and biochemical methods. Single-molecule approaches unsusceptible to these complications have led to the first glances at both compositional and conformational dynamics on the ribosome and their impact on translational control. These experiments provide the necessary link between static structure and mechanism, often providing new perspectives. Here we review recent advances in the field and their relationship to structural and biochemical data.

    View details for DOI 10.1101/cshperspect.a011551

    View details for Web of Science ID 000308739800012

    View details for PubMedID 22798542

  • Dynamics of the translational machinery CURRENT OPINION IN STRUCTURAL BIOLOGY Petrov, A., Kornberg, G., O'Leary, S., Tsai, A., Uemura, S., Puglisi, J. D. 2011; 21 (1): 137-145


    The recent growth in single molecule studies of translation has provided an insight into the molecular mechanism of ribosomal function. Single molecule fluorescence approaches allowed direct observation of the structural rearrangements occurring during translation and revealed dynamic motions of the ribosome and its ligands. These studies demonstrated how ligand binding affects dynamics of the ribosome, and the role of the conformational sampling in large-scale rearrangements intrinsic to translation elongation. The application of time-resolved cryo-electron microscopy revealed new conformational intermediates during back-translocation providing an insight into ribosomal dynamics from an alternative perspective. Recent developments permitted examination of conformational and compositional dynamics of the ribosome in real-time through multiple cycles of elongation at the single molecule level. The zero-mode waveguide approach allowed direct observation of the compositional dynamics of tRNA occupancy on the elongating ribosome. The emergence of single molecule in vivo techniques provided insights into the mechanism and regulation of translation at the organismal level.

    View details for DOI 10.1016/

    View details for Web of Science ID 000287901600017

    View details for PubMedID 21256733

  • Glycal Formation in Crystals of Uridine Phosphorylase BIOCHEMISTRY Paul, D., O'Leary, S. E., Rajashankar, K., Bu, W., Toms, A., Settembre, E. C., Sanders, J. M., Begley, T. P., Ealick, S. E. 2010; 49 (16): 3499-3509


    Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2'-deoxyuridine to 2'-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2'-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2'. Crystals of bovine uridine phosphorylase treated with 2'-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.

    View details for DOI 10.1021/bi902073b

    View details for Web of Science ID 000276817600014

    View details for PubMedID 20364833

  • Biochemical Characterization of the HpxO Enzyme from Klebsiella pneumoniae, a Novel FAD-Dependent Urate Oxidase BIOCHEMISTRY O'Leary, S. E., Hicks, K. A., Ealick, S. E., Begley, T. P. 2009; 48 (14): 3033-3035


    The HpxO enzyme from Klebsiella pneumoniae was recently proposed, on the basis of genetic studies, to catalyze the hydroxylation of uric acid to 5-hydroxyisourate as part of the purine catabolic pathway. Its primary sequence suggests that the HpxO catalytic activity depends on a flavin cofactor (FAD), contrasting with all previously studied urate oxidase enzymes, which have no cofactor requirement. Here we demonstrate biochemically that HpxO is an FAD-dependent urate oxidase. Our data are consistent with the proposal that HpxO-bound flavin hydroperoxide is the hydroxylating species. These results confirm the existence of a novel mechanistic paradigm in purine catabolism.

    View details for DOI 10.1021/bi900160b

    View details for Web of Science ID 000264983800001

    View details for PubMedID 19260710

  • O-Phospho-L-serine and the Thiocarboxylated Sulfur Carrier Protein CysO-COSH Are Substrates for CysM, a Cysteine Synthase from Mycobacterium tuberculosis BIOCHEMISTRY O'Leary, S. E., Jurgenson, C. T., Ealick, S. E., Begley, T. P. 2008; 47 (44): 11606-11615


    The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, was studied by transient-state kinetic techniques. The expression of which is upregulated under conditions of oxidative stress. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine sulfhydrylase family and employs a similar chemical mechanism involving a stable alpha-aminoacrylate intermediate. However, we show that specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine, suggesting that O-phospho-L-serine is the likely substrate in vivo. We also investigated the kinetics of the carbon-sulfur bond-forming reaction between the CysM-bound alpha-aminoacrylate intermediate and the thiocarboxylated sulfur carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than 3 orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, while the reaction with bisulfide is consistent with a rapid equilibrium binding model. We interpret this finding to suggest that the CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer. This study represents the first detailed kinetic characterization of sulfide transfer from a sulfide carrier protein.

    View details for DOI 10.1021/bi8013664

    View details for Web of Science ID 000260507100025

    View details for PubMedID 18842002