A5016740846- RNA and protein synthesis mechanisms
- Bacterial Genetics and Biotechnology
- Genomics and Phylogenetic Studies
- RNA modifications and cancer
- DNA Repair Mechanisms
- Genomics and Chromatin Dynamics
- Enzyme Structure and Function
- DNA and Nucleic Acid Chemistry
- Bacteriophages and microbial interactions
- Microbial Community Ecology and Physiology
- RNA Research and Splicing
- Advanced biosensing and bioanalysis techniques
- CRISPR and Genetic Engineering
- Photosynthetic Processes and Mechanisms
- Protist diversity and phylogeny
- Protein Structure and Dynamics
- RNA Interference and Gene Delivery
- Microbial Fuel Cells and Bioremediation
- Microbial Metabolic Engineering and Bioproduction
- Metalloenzymes and iron-sulfur proteins
- Diffusion and Search Dynamics
- Insect symbiosis and bacterial influences
- Biochemical and Molecular Research
- Polyamine Metabolism and Applications
- Algal biology and biofuel production
Colorado State University
2016-2025
The Ohio State University
2005-2014
Institute of Animal Biochemistry and Genetics of the Slovak Academy of Sciences
2008
Cornell University
2002-2007
University of Wisconsin–Madison
2003
Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report crystal structure of an archaeal histone-DNA complex. DNA wraps around extended polymer, formed by histone homodimers, quasi-continuous superhelix same geometry as nucleosome. Substitutions conserved glycine at interface adjacent protein layers destabilize chromatin, reduce growth rate, and impair transcription regulation, confirming biological importance polymeric structure. Our...
By using single-molecule measurements, we demonstrate that the elongation kinetics of individual Escherichia coli RNA polymerase molecules are remarkably homogeneous. We find no evidence distinct states among polymerases. Instead, observed heterogeneity in transcription rates results from statistical variation frequency and duration pausing. When transcribing a gene without strong pause sites, display transient pauses distributed randomly both time distance. Transitions between active mode...
Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.Sequencing cDNA libraries, generated from isolated cells growth metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map...
ABSTRACT Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis Escherichia coli have been constructed. Plasmid pTN1 from nautilis was ligated to the commercial vector pCR2.1-TOPO, markers were added so T. transformants could be selected by Δ trpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit RNA polymerase (RNAP), ∼8-fold higher...
Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed presence a second beta-glycosidase that we have identified as product TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, optimal activity at 95 degrees C from pH 8 9.5, functional half-life approximately 7 min 100 C. To generate strain with both TK1761 TK1827 deleted, new selection/counterselection protocol developed, levels in T....
Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His(6)-tagged protein known or predicted to be component the archaeal DNA replication machinery. Using proteins, stable complexes assembled in vivo isolated directly from clarified cell lysates and T. proteins present identified by mass spectrometry. Based on results obtained, network interactions among has established that confirms previously documented interactions, provides experimental...
Thermococcus kodakarensis (formerly kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter beta-glycosidase-encoding reporter gene (TK1761). Expression of the was almost fully inhibited by upstream presence 5'-TTTTTTTT (T(8)) reduced >70% intergenic sequences that contained oligo(T) sequences. An sequence (t(mcrA)) conforms bacterial intrinsic...
ABSTRACT Three evolutionarily distinct families of replicative DNA polymerases, designated polymerase B (Pol B), Pol C, and D, have been identified. Members the family are present in all three domains life, whereas C exists only Bacteria D Archaea . enzymes replicate eukaryotic chromosomal DNA, as members , it has assumed that also archaeal genomes. Here we report construction Thermococcus kodakarensis strains with mutations delete or inactivate key functions B. T. lacking had no detectable...
Thermococcus kodakarensis (T.k.) has emerged as a premier model system for studies of archaeal biochemistry, genetics, and hyperthermophily. This prominence is derived largely from the natural competence T.k. comprehensive, rapid, facile techniques available manipulation genome. These genetic capacities are complemented by robust planktonic growth, simple selections screens, defined in vitro transcription translation systems, replicative expression plasmids, vivo reporter constructs, an...
Chromosomal DNA replication requires the spatial and temporal coordination of activities several complexes that constitute replisome. A previously uncharacterized protein, encoded by TK1252 in archaeon Thermococcus kodakaraensis, was shown to stably interact with archaeal GINS complex vivo, a central component Here, we document this protein (TK1252p) is processive, single-strand DNA-specific exonuclease degrades 5′ → 3′ direction. TK1252p binds specifically GINS15 subunit T. kodakaraensis...
Abstract Background Histone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role regulating gene expression is directed by primary sequence motifs that together form code. The experiments reported were undertaken to determine if archaeal histone assembly conforms Results Eukaryotic favored phased helical repeats AA/TT/AT/TA CC/GG/CG/GC dinucleotides, disfavored longer AT-rich...
The sole unifying feature of the incredibly diverse Archaea is their isoprenoid-based ether-linked lipid membranes. Unique membrane composition, including an abundance membrane-spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding synthesis and modification lipids how dynamic changes archaeal composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl tetraethers (GDGTs), are generated by synthase (Tes)...
ABSTRACT An in vivo archaeal gene reporter system has been established based on TK1761, a that encodes nonessential β-glycosidase Thermococcus kodakaraensis . Following the introduction of nonsense codons into promoter-proximal genes, polarity operon expression this archaeon by both microarray hybridization assays and system.
Archaeal histones wrap DNA into complexes, designated archaeal nucleosomes, that resemble the tetrasome core of a eukaryotic nucleosome. Therefore, all interactions in vivo Thermococcus kodakarensis, most genetically versatile model species for research, must occur context histone-bound genome. Here we report construction and properties T. kodakarensis strains have TK1413 or TK2289 deleted, genes encode HTkA HTkB, respectively, two present this archaeon. All attempts to generate strain with...
RNA polymerase activity is regulated by nascent sequences, DNA template and conserved transcription factors. Transcription factors promoting initiation elongation have been characterized in each domain, but termination identified only bacteria eukarya. Here we describe euryarchaeal (Eta), the first archaeal factor capable of disrupting complex (TEC), detail rate requirements for Eta-mediated termination, a role Eta vivo. energy-dependent, requires upstream disrupts TECs to release solution....
Inteins (intervening proteins) interrupt host proteins and are removed through a protein splicing reaction that ligates adjacent N- C-exteins. The ability of inteins to specifically rearrange peptide bonds has proven exceptionally useful in engineering, thus, methods control intein activity considerable interest. One particularly application is for the removal an affinity tag following purification target N-terminal cleavage (NTC). Typically, extended incubation at high temperature (greater...