Author
Joseph D. Puglisi
Other affiliations: State University of New York at Fredonia, Centre national de la recherche scientifique, University of California, Berkeley ...read more
Bio: Joseph D. Puglisi is an academic researcher from Stanford University. The author has contributed to research in topics: RNA & Ribosome. The author has an hindex of 63, co-authored 192 publications receiving 14790 citations. Previous affiliations of Joseph D. Puglisi include State University of New York at Fredonia & Centre national de la recherche scientifique.
Topics: RNA, Ribosome, Transfer RNA, Translation (biology), Eukaryotic Ribosome
Papers published on a yearly basis
Papers
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TL;DR: An enzymatic oxygen scavenging system for improved dye stability in single-molecule experiments and it is found that biological reducing agents increase both the frequency and duration of blinking events of Cy5, an effect that scales with reducing potential.
794 citations
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TL;DR: The structure of the RNA-paromomycin complex explains binding of diverse aminoglycosides to the ribosome, their specific activity against prokaryotic organisms, and various resistance mechanisms, and provides insight into ribosomes function.
Abstract: Aminoglycoside antibiotics that bind to 30S ribosomal A-site RNA cause misreading of the genetic code and inhibit translocation. The aminoglycoside antibiotic paromomycin binds specifically to an RNA oligonucleotide that contains the 30S subunit A site, and the solution structure of the RNA-paromomycin complex was determined by nuclear magnetic resonance spectroscopy. The antibiotic binds in the major groove of the model A-site RNA within a pocket created by an A-A base pair and a single bulged adenine. Specific interactions occur between aminoglycoside chemical groups important for antibiotic activity and conserved nucleotides in the RNA. The structure explains binding of diverse aminoglycosides to the ribosome, their specific activity against prokaryotic organisms, and various resistance mechanisms, and provides insight into ribosome function.
778 citations
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TL;DR: This chapter discusses the experimental methods needed to acquire a melting curve and the analysis and interpretation of the data, as well as comparing results with previously published data.
Abstract: Publisher Summary This chapter discusses the experimental methods needed to acquire a melting curve and the analysis and interpretation of the data. Any standard commercial UV spectrophotometer can be equipped to measure melting curves. A useful instrument is a single-beam Gilford (Oberlin, OH) spectrophotometer (Model 2530) with an automated reference compensator that allows melting curves to be obtained on three separate samples simultaneously. One major advantage of using UV spectroscopy is the high sensitivity of the method. Normally, the absorbance of the sample used should be between 0.2 and 2.0. Sample preparation for UV melting studies is straightforward. The RNA stock solution is prepared by dialysis against the desired buffer and different concentrations are made by dilution. The high salt concentration is chosen to minimize electrostatic repulsion between strands and to avoid divalent ions, which catalyze hydrolysis of RNA and favor triple-strand formation. This solvent provides a standard condition for measuring melting curves and for comparing results with previously published data.
613 citations
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TL;DR: The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-n nucleotide loop and a three-nucleotide bulge that stabilizes arginine hydrogen bonding to G26 and phosphates.
Abstract: The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-nucleotide loop and a three-nucleotide bulge. The conformations of TAR RNA and of TAR with an arginine analog specifically bound at the binding site for the viral protein, Tat, were characterized by nuclear magnetic resonance (NMR) spectroscopy. Upon arginine binding, the bulge changes conformation, and essential nucleotides for binding, U23 and A27.U38, form a base-triple interaction that stabilizes arginine hydrogen bonding to G26 and phosphates. Specificity in the arginine-TAR interaction appears to be derived largely from the structure of the RNA.
581 citations
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TL;DR: Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation, suggesting that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states.
Abstract: Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by approximately 6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components.
492 citations
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28 Jul 2005
TL;DR: PfPMP1)与感染红细胞、树突状组胞以及胎盘的单个或多个受体作用,在黏附及免疫逃避中起关键的作�ly.
Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1(PfPMP1)与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用,在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员,通过启动转录不同的var基因变异体为抗原变异提供了分子基础。
18,940 citations
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TL;DR: This work describes a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes, which can be programmed to bear complex patterns such as words and images on their surfaces.
Abstract: 'Bottom-up fabrication', which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex).
6,141 citations
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TL;DR: Single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs) are presented.
Abstract: We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.
3,346 citations
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TL;DR: The crystal structure of the large ribosomal subunit from Haloarcula marismortui is determined at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins.
Abstract: The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior.
3,266 citations
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TL;DR: Anion recognition chemistry has grown from its beginnings with positively charged ammonium cryptand receptors for halide binding to a plethora of charged and neutral, cyclic and acyclic, inorganic and organic supramolecular host systems for the selective complexation, detection, and separation of anionic guest species.
Abstract: Anion recognition chemistry has grown from its beginnings in the late 1960s with positively charged ammonium cryptand receptors for halide binding to, at the end of the millennium, a plethora of charged and neutral, cyclic and acyclic, inorganic and organic supramolecular host systems for the selective complexation, detection, and separation of anionic guest species. Solvation effects and pH values have been shown to play crucial roles in the overall anion recognition process. More recent developments include exciting advances in anion-templated syntheses and directed self-assembly, ion-pair recognition, and the function of anions in supramolecular catalysis.
3,145 citations