Younggyu Kim

Transcription Activation at Class II CAP-Dependent Promoters: Two Interactions between CAP and RNA Polymerase

At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase. We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain. We further show that the two interactions affect different steps in transcription initiation. Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence.

Structural Organization of the RNA Polymerase-Promoter Open Complex

We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.

Efficient Site-Specific Labeling of Proteins via Cysteines

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the proteins amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the proteins function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Intermediate Waters in the East/Japan Sea

Properties of the intermediate layer in the East/Japan Sea are examined by using CREAMS data taken mainly in summer of 1995. Vertical profiles of potential temperature, salinity and dissolved oxygen and relationships between these physical and chemical properties show that the dissolved oxygen concentration of 250 µmol/l, roughly corresponding to 0.6°C at the depth of about 400 db, makes a boundary between intermediate and deep waters. Water colder than 0.6°C has a very stable relationship between potential temperature and salinity while salinity of the water warmer than 0.6°C is lower in the western Japan Basin than that in the eastern Japan Basin. The low salinity water with high oxygen corresponds to the East Sea Intermediate Water (ESIW; <34.06 psu, >250 µmol/l and >1.0°C) which was previously identified by Kim and Chung (1984) and the high salinity water with high oxygen found in eastern Japan Basin is named as the High Salinity Intermediate Water (HSIW; >34.07 psu, >250 µmol/l and >0.6°C). Spatial distribution of salinity and acceleration potential on the surface of σϑ = 27.2 kg/m3 shows that the ESIW prevailing in the western Japan Basin is transported eastward by a zonal flow along the polar front near 40°N and a cyclonic gyre in the eastern Japan Basin is closely related to the HSIW.

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions −78 to +20 of a Class I CAP-dependent promoter with a CAP site at position −61.5 and a premelted transcription bubble. A 20-Å electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP α subunit C-terminal domain (αCTD), interactions of αCTD with σ70 region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP β′, β, and σ70 subunits.

Preparation of non-aggregated fluorescent nanodiamonds (FNDs) by non-covalent coating with a block copolymer and proteins for enhancement of intracellular uptake

Fluorescent nanodiamonds (FNDs) are very promising fluorophores for use in biosystems due to their high biocompatibility and photostability. To overcome their tendency to aggregate in physiological solutions, which severely limits the biological applications of FNDs, we developed a new non-covalent coating method using a block copolymer, PEG-b-P(DMAEMA-co-BMA), or proteins such as BSA and HSA. By simple mixing of the block copolymer with FNDs, the cationic DMAEMA and hydrophobic BMA moieties can strongly interact with the anionic and hydrophobic moieties on the FND surface, while the PEG block can form a shell to prevent the direct contact between FNDs. The polymer-coated FNDs, along with BSA- and HSA-coated FNDs, showed non-aggregation characteristics and maintained their size at the physiological salt concentration. The well-dispersed, polymer- or protein-coated FNDs in physiological solutions showed enhanced intracellular uptake, which was confirmed by CLSM. In addition, the biocompatibility of the coated FNDs was expressly supported by a cytotoxicity assay. Our simple non-covalent coating with the block copolymer, which can be easily modified by various chemical methods, projects a very promising outlook for future biomedical applications, especially in comparison with covalent coating or protein-based coating.

Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13.

Abstract Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors.

Toward Single-Molecule Optical Mapping of the Epigenome

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical-based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nanoscale and enhanced optical resolving power for analysis of genomic information. During the past few years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.

Electrochemical property of surface modified polypyrrole film with heteropoly anions

Summary form only given. Preparation of heteropoly anion modified polypyrrole on glassy carbon and their electrochemical properties were studied. Keggin-type heteropolymolybdate and heteropolytungstate were chosen in this study because of their distinct catalytic activity towards the reduction of oxygen and nitrite in the solution. Electrochemical polymerization and incorporation of poly-anions were performed in solution containing both chemicals. The first scan of cyclic voltammogram showed the polymer film grew via nucleation and growth mechanism. Electrochemical peaks from the film were from polymer itself and from the incorporated poly anions. They were similar to those obtained from the solution and were stable under pH value of 4.5. The peak potential due to polypyrrole shifted to cathodic direction by about 40 mV/pH, while those of poly anions peak exhibited cathodic shift of about 60 mV/pH. As the thickness of the film changed the charge transport mechanism also changed and the redox peaks shifted to cathodic direction. The amount of incorporated anions determined by electrochemical method linearly depended on the charge passed in the polymerization process. The apparent diffusion coefficient of incorporated poly-anions in the polymer film was determined to be in the order of 10/sup -9/ to 10/sup -10/ cm/sup 2//sec. Electrochemical quartz crystal microbalance technique provided some insight of the film formation and the incorporation of poly anion. Also the catalytic activity towards the reduction of oxygen and hydrogen peroxide will be discussed.

Rapid and facile synthesis of a (ZnxAgyInz)S2 nanocrystal library via sono-combichem method and its characterization including single nanocrystal analysis

We have developed a facile and rapid synthesis of a highly fluorescent nanocrystal (NC) library, based on ultrasonic and combinatorial chemistry. Among a total of 66 tZAIS ((ZnxAgyInz)S2) NCs, many NCs are highly fluorescent upon UV irradiation, and are categorized into blue, green, yellow, orange, and red by their distinct emission wavelength ranges. The tZAIS NCs not only have long fluorescence lifetimes, but also show comparable or even higher brightness than QDs without photoblinking in single NC analysis. Their unique photophysical properties together with less-toxic nature can permit the tZAIS system to be practically utilized in various bioimaging fields, especially single nanoparticle-based imaging and tracking.