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All Publications

  • High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs. Nature communications Ha, B., Larsen, K. P., Zhang, J., Fu, Z., Montabana, E., Jackson, L. N., Chen, D., Puglisi, E. V. 2021; 12 (1): 2500


    Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9A, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

    View details for DOI 10.1038/s41467-021-22628-9

    View details for PubMedID 33947853

  • Advances in understanding the initiation of HIV-1 reverse transcription. Current opinion in structural biology Krupkin, M. n., Jackson, L. N., Ha, B. n., Puglisi, E. V. 2020; 65: 175?83


    Many viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Human Immunodeficiency Virus (HIV), use RNA as their genetic material. How viruses harness RNA structure and RNA-protein interactions to control their replication remains obscure. Recent advances in the characterization of HIV-1 reverse transcriptase, the enzyme that converts its single-stranded RNA genome into a double-stranded DNA copy, reveal how the reverse transcription complex evolves during initiation. Here we highlight these advances in HIV-1 structural biology and discuss how they are furthering our understanding of HIV and related ribonucleoprotein complexes implicated in viral disease.

    View details for DOI 10.1016/

    View details for PubMedID 32916568

  • Distinct Conformational States Underlie Pausing during Initiation of HIV-1 Reverse Transcription. Journal of molecular biology Larsen, K. P., Choi, J. n., Jackson, L. N., Kappel, K. n., Zhang, J. n., Ha, B. n., Chen, D. H., Puglisi, E. V. 2020


    A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have identified specific sites along the viral RNA genomic template in which reverse transcriptase (RT) stalls. These stalling points, which occur after the addition of 3 and 5 template dNTPs, may serve as checkpoints to regulate the precise timing of HIV-1 reverse transcription following viral entry. Structural studies of reverse transcriptase initiation complexes (RTICs) have revealed unique conformations that may explain the slow rate of incorporation, however, questions remain about the temporal evolution of the complex and features that contribute to strong pausing during initiation. Here we present cryo-electron microscopy (cryo-EM) and single-molecule characterization of an RTIC after three rounds of dNTP incorporation (+3), the first major pausing point during reverse transcription initiation. Cryo-EM structures of a?+?3 extended RTIC reveal conformational heterogeneity within the RTIC core. Three distinct conformations were identified, two of which adopt unique, likely off-pathway, intermediates in the canonical polymerization cycle. Single-molecule F÷rster resonance energy transfer (smFRET) experiments confirm that the +3 RTIC is more structurally dynamic than earlier stage RTICs. These alternative conformations were selectively disrupted through structure-guided point mutations to shift smFRET populations back towards the on-pathway conformation. Our results support the hypothesis that conformational heterogeneity within the HIV-1 reverse transcriptase initiation complex during pausing serves as an additional means of regulating HIV-1 replication.

    View details for DOI 10.1016/j.jmb.2020.06.003

    View details for PubMedID 32512005

  • Relating Structure and Dynamics in RNA Biology. Cold Spring Harbor perspectives in biology Larsen, K. P., Choi, J., Prabhakar, A., Puglisi, E. V., Puglisi, J. D. 2019; 11 (7)


    SUMMARYRecent advances in structural biology methods have enabled a surge in the number of RNA and RNA-protein assembly structures available at atomic or near-atomic resolution. These complexes are often trapped in discrete conformational states that exist along a mechanistic pathway. Single-molecule fluorescence methods provide temporal resolution to elucidate the dynamic mechanisms of processes involving complex RNA and RNA-protein assemblies, but interpretation of such data often requires previous structural knowledge. Here we highlight how single-molecule tools can directly complement structural approaches for two processes--translation and reverse transcription-to provide a dynamic view of molecular function.

    View details for DOI 10.1101/cshperspect.a032474

    View details for PubMedID 31262948

  • Expanding single-molecule fluorescence spectroscopy to capture complexity in biology. Current opinion in structural biology Choi, J., Grosely, R., Puglisi, E. V., Puglisi, J. D. 2019


    Fundamental biological processes are driven by diverse molecular machineries. In recent years, single-molecule fluorescence spectroscopy has matured as a unique tool in biology to study how structural dynamics of molecular complexes drive various biochemical reactions. In this review, we highlight underlying developments in single-molecule fluorescence methods that enable deep biological investigations. Recent progress in these methods points toward increasing complexity of measurements to capture biological processes in a living cell, where multiple processes often occur simultaneously and are mechanistically coupled.

    View details for DOI 10.1016/

    View details for PubMedID 31213390

  • De novo computational RNA modeling into cryo-EM maps of large ribonucleoprotein complexes. Nature methods Kappel, K., Liu, S., Larsen, K. P., Skiniotis, G., Puglisi, E. V., Puglisi, J. D., Zhou, Z. H., Zhao, R., Das, R. 2018


    Increasingly, cryo-electron microscopy (cryo-EM) is used to determine the structures of RNA-protein assemblies, but nearly all maps determined with this method have biologically important regions where the local resolution does not permit RNA coordinate tracing. To address these omissions, we present de novo ribonucleoprotein modeling in real space through assembly of fragments together with experimental density in Rosetta (DRRAFTER). We show that DRRAFTER recovers near-native models for a diverse benchmark set of RNA-protein complexes including the spliceosome, mitochondrial ribosome, and CRISPR-Cas9-sgRNA complexes; rigorous blind tests include yeast U1 snRNP and spliceosomal P complex maps. Additionally, to aid in model interpretation, we present a method for reliable in situ estimation of DRRAFTER model accuracy. Finally, we apply DRRAFTER to recently determined maps of telomerase, the HIV-1 reverse transcriptase initiation complex, and the packaged MS2 genome, demonstrating the acceleration of accurate model building in challenging cases.

    View details for PubMedID 30377372

  • Dynamic Interplay of RNA and Protein in the Human Immunodeficiency Virus-1 Reverse Transcription Initiation Complex. Journal of molecular biology Coey, A. T., Larsen, K. P., Choi, J., Barrero, D. J., Puglisi, J. D., Puglisi, E. V. 2018


    The initiation of reverse transcription in human immunodeficiency virus-1 (HIV-1) is a key early step in the virus replication cycle. During this process, the viral enzyme reverse transcriptase (RT) copies the single-stranded viral RNA (vRNA) genome into double-stranded DNA using human tRNALys3 as a primer for initiation. The tRNA primer and vRNA genome contain several complementary sequences that are important for regulating reverse transcription initiation kinetics. Using single-molecule Forster resonance energy transfer (smFRET) spectroscopy, we demonstrate that the vRNA-tRNA initiation complex is conformationally heterogeneous and dynamic in the absence of RT. As shown previously, nucleic acid-RT interaction is characterized by rapid dissociation constants. We show that extension of the vRNA-tRNA primer binding site (PBS) helix from 18 base pairs to 22 base pairs stabilizes RT binding to the complex and that the tRNA 5' end has a role in modulating RT binding. RT occupancy on the complex stabilizes helix 1 (H1) formation and reduces global structural heterogeneity. The stabilization of H1 upon RT binding may serve to destabilize helix 2 (H2), the first pause site for RT during initiation, during later steps of reverse transcription initiation.

    View details for PubMedID 30201267

  • Single-Molecule Fluorescence Applied to Translation. Cold Spring Harbor perspectives in biology Prabhakar, A., Puglisi, E. V., Puglisi, J. D. 2018


    Single-molecule fluorescence methods have illuminated the dynamics of the translational machinery. Structural and bulk biochemical experiments have provided detailed atomic and global mechanistic views of translation, respectively. Single-molecule studies of translation have bridged these views by temporally connecting the conformational and compositional states defined from structural data within the mechanistic framework of translation produced from biochemical studies. Here, we discuss the context for applying different single-molecule fluorescence experiments, and present recent applications to studying prokaryotic and eukaryotic translation. We underscore the power of observing single translating ribosomes to delineate and sort complex mechanistic pathways during initiation and elongation, and discuss future applications of current and improved technologies.

    View details for PubMedID 29891562

  • Architecture of an HIV-1 reverse transcriptase initiation complex. Nature Larsen, K. P., Mathiharan, Y. K., Kappel, K. n., Coey, A. T., Chen, D. H., Barrero, D. n., Madigan, L. n., Puglisi, J. D., Skiniotis, G. n., Puglisi, E. V. 2018


    Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection 1 and a common target of antiretroviral drugs 2 . The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA 5 each boundáto host lysine 3 transfer RNA (tRNALys3), which acts as a primer for initiation of reverse transcription6,7. Upon viral entry into cells, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5' end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

    View details for PubMedID 29695867

  • Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site RNA Coey, A., Larsen, K., Puglisi, J. D., Puglisi, E. V. 2016; 22 (11): 1689-1698


    Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5' UTR of the HIV genomic RNA (vRNA) and a host primer tRNA(Lys)3 Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.

    View details for DOI 10.1261/rna.056804.116

    View details for PubMedID 27613581

  • Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Navon, S. P., Kornberg, G., Chen, J., Schwartzman, T., Tsai, A., Puglisi, E. V., Puglisi, J. D., Adir, N. 2016; 113 (26): 7166-7170


    Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

    View details for DOI 10.1073/pnas.1606518113

    View details for PubMedID 27307442

  • Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II NATURE METHODS Sierra, R. G., Gati, C., Laksmono, H., Dao, E. H., Gul, S., Fuller, F., Kern, J., Chatterjee, R., Ibrahim, M., Brewster, A. S., Young, I. D., Michels-Clark, T., Aquila, A., Liang, M., Hunter, M. S., Koglin, J. E., Boutet, S., Junco, E. A., Hayes, B., Bogan, M. J., Hampton, C. Y., Puglisi, E. V., Sauter, N. K., Stan, C. A., Zouni, A., Yano, J., Yachandra, V. K., Soltis, S. M., Puglisi, J. D., DeMirci, H. 2016; 13 (1): 59-?


    We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-┼ structure), Thermosynechococcus elongatus photosystem II (<3-┼ diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-┼ structure).

    View details for DOI 10.1038/NMETH.3667

    View details for Web of Science ID 000367463600028

    View details for PubMedCentralID PMC4890631

  • The molecular choreography of protein synthesis: translational control, regulation, and pathways. Quarterly reviews of biophysics Chen, J. n., Choi, J. n., O'Leary, S. E., Prabhakar, A. n., Petrov, A. n., Grosely, R. n., Puglisi, E. V., Puglisi, J. D. 2016; 49: e11


    Translation of proteins by the ribosome regulates gene expression, with recent results underscoring the importance of translational control. Misregulation of translation underlies many diseases, including cancer and many genetic diseases. Decades of biochemical and structural studies have delineated many of the mechanistic details in prokaryotic translation, and sketched the outlines of eukaryotic translation. However, translation may not proceed linearly through a single mechanistic pathway, but likely involves multiple pathways and branchpoints. The stochastic nature of biological processes would allow different pathways to occur during translation that are biased by the interaction of the ribosome with other translation factors, with many of the steps kinetically controlled. These multiple pathways and branchpoints are potential regulatory nexus, allowing gene expression to be tuned at the translational level. As research focus shifts toward eukaryotic translation, certain themes will be echoed from studies on prokaryotic translation. This review provides a general overview of the dynamic data related to prokaryotic and eukaryotic translation, in particular recent findings with single-molecule methods, complemented by biochemical, kinetic, and structural findings. We will underscore the importance of viewing the process through the viewpoints of regulation, translational control, and heterogeneous pathways.

    View details for PubMedID 27658712

  • 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


    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 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


    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


    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 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


    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 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


    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 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


    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 PubMedID 18079727

  • NMR investigation of HIV reverse transcription initiation 4th NATO Advanced-Study-Institute on Dynamics, Structure and Function of Biological Macromolecules Puglisi, E. V. I O S PRESS. 2001: 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


    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 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


    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

  • Structure and function of ribosomal RNA International Conference on the Structure and Function of the Ribosome 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


    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



    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



    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 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


    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


    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


    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

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