Bio

Academic Appointments


Honors & Awards


  • Graduate Fellowship, Italian Ministry of Education and Scientific Research (1991-1993)
  • Fellowhip for University Research, Italian Pharmacy Consurtium (1989-1990)
  • Fellowship for University Research, European Economic Community (1988)

Professional Education


  • Ph.D., University of Brescia, Italy, Microbiology (1994)
  • Dott., University of Parma, Italy, Chemistry (1989)
  • Laurea, University of Parma, Italy, Chemistry (1985)

Community and International Work


  • Int'l School of Biological Magnetic Resonance, 10th Course, 22 June-2 July 2010, Erice-Sicily, Italy

    Topic

    NMR; Biophysics; Structural Biology; Biochemistry; X-ray Crystallography; Computational Biology

    Partnering Organization(s)

    NATO; NSF; EMFCSC; Russian Academy of Sciences

    Populations Served

    Graduate Students and Postdoctoral Scholars

    Location

    International

    Ongoing Project

    No

    Opportunities for Student Involvement

    Yes

  • Int'l School of Biological Magnetic Resonance, 9th Course, 22 June-2 July 2009, Erice-Sicily, Italy

    Topic

    NMR; Biophysics; Structural Biology; Biochemistry; X-ray Crystallography; Computational Biology

    Partnering Organization(s)

    NATO; NSF; EMFCSC; Russian Academy of Sciences

    Populations Served

    Graduate Students and Postdoctoral Fellows

    Location

    International

    Ongoing Project

    No

    Opportunities for Student Involvement

    Yes

Research & Scholarship

Current Research and Scholarly Interests


Viral infections and subsequent host response depend on multiple RNA-protein interaction. My research focuses on the structural and functional characterization of RNA-protein complexes involved in viral infection. Current research aims to understand how the Human Immunodeficiency Virus (HIV) initiates its replication upon host infection. We use NMR spectroscopy and x-ray crystallography to study the structure of the initiation complex, formed by a host tRNA and HIV genomic RNA, coupled with biochemical and biophysical methods to understand functional properties. The goal of this research is to gain a molecular view of HIV replication initiation, and use this information to develop new therapeutic approaches to combat HIV.

Teaching

2013-14 Courses


Publications

Journal Articles


  • The Impact of Aminoglycosides on the Dynamics of Translation Elongation CELL REPORTS Tsai, A., Uemura, S., Johansson, M., Puglisi, E. V., Marshall, R. A., Aitken, C. E., Korlach, J., Ehrenberg, M., Puglisi, J. D. 2013; 3 (2): 497-508

    Abstract

    Inferring antibiotic mechanisms on translation through static structures has been challenging, as biological systems are highly dynamic. Dynamic single-molecule methods are also limited to few simultaneously measurable parameters. We have circumvented these limitations with a multifaceted approach to investigate three structurally distinct aminoglycosides that bind to the aminoacyl-transfer RNA site (A site) in the prokaryotic 30S ribosomal subunit: apramycin, paromomycin, and gentamicin. Using several single-molecule fluorescence measurements combined with structural and biochemical techniques, we observed distinct changes to translational dynamics for each aminoglycoside. While all three drugs effectively inhibit translation elongation, their actions are structurally and mechanistically distinct. Apramycin does not displace A1492 and A1493 at the decoding center, as demonstrated by a solution nuclear magnetic resonance structure, causing only limited miscoding; instead, it primarily blocks translocation. Paromomycin and gentamicin, which displace A1492 and A1493, cause significant miscoding, block intersubunit rotation, and inhibit translocation. Our results show the power of combined dynamics, structural, and biochemical approaches to elucidate the complex mechanisms underlying translation and its inhibition.

    View details for DOI 10.1016/j.celrep.2013.01.027

    View details for Web of Science ID 000321895200022

    View details for PubMedID 23416053

  • RNA purification by preparative polyacrylamide gel electrophoresis. Methods in enzymology Petrov, A., Wu, T., Puglisi, E. V., Puglisi, J. D. 2013; 530: 315-330

    Abstract

    Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length. It is commonly used to separate and purify RNA species after in vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to remove degradation products, and to purify labeled RNA species. To preserve RNA integrity following purification, RNA is usually visualized by UV shadowing or stained with ethidium bromide or SYBR green dyes.

    View details for DOI 10.1016/B978-0-12-420037-1.00017-8

    View details for PubMedID 24034329

  • Secondary Structure of the HIV Reverse Transcription Initiation Complex by NMR JOURNAL OF MOLECULAR BIOLOGY Puglisi, E. V., Puglisi, J. D. 2011; 410 (5): 863-874

    Abstract

    Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus (HIV) upon infection of a host cell. Viral reverse transcriptase initiates from a specific RNA-RNA complex formed between a host transfer RNA (tRNA(Lys)(3)) and a region at the 5' end of genomic RNA; the 3' end of the tRNA acts as a primer for reverse transcription of genomic RNA. We report here the secondary structure of the HIV genomic RNA-human tRNA(Lys)(3) initiation complex using heteronuclear nuclear magnetic resonance methods. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of the 18-bp primer helix with the 3' end of tRNA(Lys)(3) drives large conformational rearrangements of the tRNA at the 5' end while maintaining the anticodon loop for potential loop-loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse transcription.

    View details for DOI 10.1016/j.jmb.2011.04.024

    View details for Web of Science ID 000293674100007

    View details for PubMedID 21763492

  • Probing the conformation of human tRNA(3)(Lys) in solution by NMR FEBS LETTERS Puglisi, E. V., Puglisi, J. D. 2007; 581 (27): 5307-5314

    Abstract

    Human tRNA(3)(Lys) acts as a primer for the reverse transcription of human immunodeficiency virus genomic RNA. To form an initiation complex with genomic RNA, tRNA(3)(Lys) must reorganize its secondary structure. To provide a starting point for mechanistic studies of the formation of the initiation complex, we here present solution NMR investigations of human tRNA(3)(Lys). We use a straightforward set of NMR experiments to show that tRNA(3)(Lys) adopts a standard transfer ribonucleic acid tertiary structure in solution, and that Mg(2+) is required for this folding. The results underscore the power of NMR to reveal rapidly the conformation of RNAs.

    View details for DOI 10.1016/j.febslet.2007.10.026

    View details for Web of Science ID 000253487700022

    View details for PubMedID 17963705

  • Rapid purification of RNAs using fast performance liquid chromatography (FPLC) RNA-A PUBLICATION OF THE RNA SOCIETY Kim, I., Mckenna, S. A., Puglisi, E. V., Puglisi, J. D. 2007; 13 (2): 289-294

    Abstract

    We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.

    View details for DOI 10.1261/rna.342607

    View details for Web of Science ID 000243753500011

    View details for PubMedID 17179067

  • Purification and characterization of transcribed RNAs using gel filtration chromatography NATURE PROTOCOLS Mckenna, S. A., Kim, I., Puglisi, E. V., Lindhout, D. A., Aitken, C. E., Marshall, R. A., Puglisi, J. D. 2007; 2 (12): 3270-3277

    Abstract

    RNA synthesis using in vitro transcription by phage T7 RNA polymerase allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. Previous purification approaches relied on gel electrophoretic or gravity-flow chromatography methods. We present here a protocol for the in vitro transcription of RNAs and subsequent purification using fast-performance liquid chromatography. This protocol greatly facilitates production of RNA in a single day from transcription to purification.

    View details for DOI 10.1038/nprot.2007.480

    View details for Web of Science ID 000253140200026

    View details for PubMedID 18079727

  • NMR investigation of HIV reverse transcription initiation DYNAMICS, STRUCTURE AND FUNCTION OF BIOLOGICAL MACROMOLECULES Puglisi, E. V. 2001; 315: 187-192
  • HIV-1 A-rich RNA loop mimics the tRNA anticodon structure NATURE STRUCTURAL BIOLOGY Puglisi, E. V., Puglisi, J. D. 1998; 5 (12): 1033-1036

    Abstract

    Interaction of HIV-1 genomic RNA and human tRNA(Lys)3 initiates viral reverse transcription. An adenosine-rich (A-rich) loop in HIV RNA mediates complex formation between tRNA and viral RNA. We have determined the structure of an A-rich loop oligonucleotide using nuclear magnetic resonance spectroscopy. The loop structure is stabilized by a noncanonical G-A pair and a U-turn motif, which leads to stacking of the conserved adenosines. The structure has similarity to the tRNA anticodon structure, and suggests possible mechanisms for its role in initiation of reverse transcription.

    View details for Web of Science ID 000077284900005

    View details for PubMedID 9846871

  • Structure of a conserved RNA component of the peptidyl transferase centre NATURE STRUCTURAL BIOLOGY Puglisi, E. V., Green, R., Noller, H. F., Puglisi, J. D. 1997; 4 (10): 775-778

    Abstract

    The structure of a conserved hairpin loop involved in peptidyl-tRNA recognition by 50S ribosomal subunits has been solved by NMR. The loop is closed by a novel G-C base pair and presents guanine residues for RNA recognition.

    View details for Web of Science ID A1997YA20300005

    View details for PubMedID 9334738

  • NMR ANALYSIS OF TRANSFER-RNA ACCEPTOR STEM MICROHELICES - DISCRIMINATOR BASE CHANGE AFFECTS TRANSFER-RNA CONFORMATION AT THE 3' END PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Puglisi, E. V., Puglisi, J. D., Williamson, J. R., RajBhandary, U. L. 1994; 91 (24): 11467-11471

    Abstract

    An important step in initiation of protein synthesis in Escherichia coli is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Here we use NMR spectroscopy to characterize the conformation of two RNA microhelices, which correspond to the acceptor stem of mutants of E. coli initiator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs. One of the mutant tRNAs is an extremely poor substrate for Met-tRNA transformylase, whereas the other one is a much better substrate. We show that one microhelix forms a structure in which its 3'-ACCA sequence extends the stacking of the acceptor stem. The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in close proximity to G1. These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' end. They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA transformylase.

    View details for Web of Science ID A1994PU28500038

    View details for PubMedID 7972085

  • A MONOCLONAL-ANTIBODY TO THE NH2-TERMINAL SEGMENT OF HUMAN IFN-GAMMA SELECTIVELY INTERFERES WITH THE ANTIPROLIFERATIVE ACTIVITY OF THE LYMPHOKINE JOURNAL OF IMMUNOLOGY Caruso, A., Tiberio, L., DERANGO, C., Bonfanti, C., FLAMMINIO, G., Gribaudo, G., Monti, E., Viani, E., Manca, N., Garotta, G., Landolfo, S., Balsari, A., Turano, A. 1993; 150 (3): 1029-1035

    Abstract

    To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.

    View details for Web of Science ID A1993KJ09500034

    View details for PubMedID 8423329

  • Molecular Recognition in Water: New Receptors for Academic Derivatives J. Am. Chem. Soc. Rotello, V., Viani E., Deslongchamps G, Murray BA., Rebek J Jr. 1993: 797-798
  • NATURAL HUMAN-ANTIBODIES TO GAMMA INTERFERON INTERFERE WITH THE IMMUNOMODULATING ACTIVITY OF THE LYMPHOKINE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Turano, A., Balsari, A., Viani, E., Landolfo, S., Zanoni, L., Gargiulo, F., Caruso, A. 1992; 89 (10): 4447-4451

    Abstract

    Natural antibodies to gamma interferon (IFN-gamma) were found in patients suffering from different viral diseases and, at a lower titer, in healthy individuals. Such antibodies were affinity-purified and studied for their capability to interfere in vitro with the antiviral and immunomodulating activity of IFN-gamma. Data obtained show that these human anti-IFN-gamma antibodies have no inhibitory effect on the antiviral activity of IFN-gamma. On the contrary, they are able to inhibit the expression of Fc receptor sites and HLA-DR antigens induced by IFN-gamma on the U-937 cells, a human monocytoid/macrophage-derived cell line. These antibodies can also interfere in a mixed lymphocyte culture (MLC) with the proliferation of lymphocytes and the generation of cytotoxic lymphocytes. However, they showed only a moderate inhibitory effect on the cytotoxicity generated in MLC to K-562 cells. Human antibodies capable of interfering with the immunomodulating activities of IFN-gamma might open up a new field in clinical therapy for those diseases that carry evidence of activated cell-mediated immunity.

    View details for Web of Science ID A1992HU97700047

    View details for PubMedID 1316609

  • INHIBITION OF THE BIOLOGICAL-ACTIVITY OF HUMAN INTERFERON-GAMMA BY ANTIPEPTIDE ANTIBODIES JOURNAL OF INTERFERON RESEARCH Caruso, A., Viani, E., Tiberio, L., Pollara, P., Monti, E., Bonfanti, C., Gao, J., Landolfo, S., Balsari, A., Turano, A. 1992; 12 (1): 49-54

    Abstract

    To study the domain(s) responsible for the different biological activities of human interferon-gamma (HuIFN-gamma), rabbits were immunized with peptides corresponding to the five most hydrophilic amino acid sequences of the lymphokine. The resulting antisera were able to recognize both the immunizing peptide and the native protein. Antibodies to the carboxy-terminal region (amino acids 125-137) of HuIFN-gamma were able to interfere with the immunomodulating, antiviral, and antiproliferative activities of the lymphokine. Inhibition of the antiproliferative and antiviral activity was also observed using antibodies raised against the amino-terminal region (amino acids 4-16) and amino acids 80-95, respectively, but to a lesser extent than that observed with antibodies to the carboxyl terminus. The capability of these antibody preparations to partially interfere with only one of the lymphokine's biological activities might be explained by a mechanism of steric hindrance. The use of polyclonal antibodies allowed us to limit the presence of epitopes responsible for recombinant (r)HuIFN-gamma biological activities to the carboxy-terminal region.

    View details for Web of Science ID A1992HE76000008

    View details for PubMedID 1573282

  • PURIFICATION OF NATURAL HUMAN IFN-GAMMA ANTIBODIES IMMUNOLOGY LETTERS Viani, E., FLAMMINIO, G., Caruso, A., Foresti, I., DeFrancesco, M., Pollara, P., Balsari, A., Turano, A. 1991; 30 (1): 53-58

    Abstract

    Natural antibodies to interferon gamma (IFN-gamma) were found in patients suffering from various viral infections, but also at weak titers in healthy individuals. In the present study we describe a one-step chromatographic procedure for the purification of the anti-IFN-gamma antibodies from human Ig preparations, using a recombinant IFN-gamma-coupled Sepharose CL4B affinity column. The antibodies to IFN-gamma were eluted from the column using 3 different methods without loss of immunological activity. They were found to be Ig, mostly of the IgG1 subclass, and, in the biological assay, to be able to neutralize the de novo expression of Fc receptor sites induced by IFN-gamma on U937 cells.

    View details for Web of Science ID A1991GJ90300009

    View details for PubMedID 1959942

  • NATURAL ANTIBODIES TO IFN-GAMMA IN MAN AND THEIR INCREASE DURING VIRAL-INFECTION JOURNAL OF IMMUNOLOGY Caruso, A., Bonfanti, C., Colombrita, D., DeFrancesco, M., DERANGO, C., Foresti, I., Gargiulo, F., Gonzales, R., Gribaudo, G., Landolfo, S., Manca, N., Manni, M., Pirali, F., Pollara, P., Ravizzola, G., SCURA, G., Terlenghi, L., Viani, E., Turano, A. 1990; 144 (2): 685-690

    Abstract

    Natural antibodies to IFN-gamma were found in healthy individuals ranging from newborn babies to adults and, at higher levels, in patients suffering from different viral infections. During a viral infection, the titer of anti-IFN-gamma antibodies was observed to be correlated with the stage of the disease. Antibodies specific to IFN-gamma were affinity purified both from sera taken from healthy individuals and sera from viral-infected patients, by using a rIFN-gamma-coupled CNBr-activated Sepharose 4B column. The antibodies were found to be of the IgG class, and maintained their ability to bind rIFN-gamma. They were then tested for neutralizing activity and none of the IgG preparations we analyzed impaired the antiviral activity of rIFN-gamma. This finding suggests that the antigenic determinants recognized by these antibodies on the IFN-gamma molecule are located outside the site, on the IFN-gamma molecule, responsible for its antiviral activity.

    View details for Web of Science ID A1990CH29500041

    View details for PubMedID 1688583

Conference Proceedings


  • Structure and function of ribosomal RNA Noller, H. F., Green, R., HEILEK, G., HOFFARTH, V., Huttenhofer, A., Joseph, S., Lee, I., Lieberman, K., Mankin, A., Merryman, C., Powers, T., Puglisi, E. V., Samaha, R. R., Weiser, B. CANADIAN SCIENCE PUBLISHING, NRC RESEARCH PRESS. 1995: 997-1009

    Abstract

    A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe(2+)-EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+ tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3'-biotinderivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+ tethered to the 5' end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the alpha-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson-Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson-Crick base pair.

    View details for Web of Science ID A1995UE60300027

    View details for PubMedID 8722015

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