Mini-Symposium: Best practices for REU programs and UNL

If you want to learn how to run an REU (Research Experience for Undergraduates) program (or to make your REU program run more smoothly), then this is your opportunity to ask questions. The presentation will begin with 5-min talks from five REU program coordinators. These coordinators run a wide range of programs and have experience ranging from 1 year to 12 years. They will mention things such as how many applicants their program receives, how they review those files, an outline of the non-research portion of their summer program, and what sorts of outcomes they collect so they can report them to NSF. Then, the coordinators will form a panel to answer questions. Shannon Bartelt-Hunt, Department of Civil Engineering Mark Griep, Department of Chemistry Don Becker, Redox Biolog

Mini-Symposium: Best practices for REU programs and UNL

If you want to learn how to run an REU (Research Experience for Undergraduates) program (or to make your REU program run more smoothly), then this is your opportunity to ask questions. The presentation will begin with 5-min talks from five REU program coordinators. These coordinators run a wide range of programs and have experience ranging from 1 year to 12 years. They will mention things such as how many applicants their program receives, how they review those files, an outline of the non-research portion of their summer program, and what sorts of outcomes they collect so they can report them to NSF. Then, the coordinators will form a panel to answer questions. Shannon Bartelt-Hunt, Department of Civil Engineering Mark Griep, Department of Chemistry Don Becker, Redox Biolog

Mini-Symposium: Best practices for REU programs and UNL

If you want to learn how to run an REU (Research Experience for Undergraduates) program (or to make your REU program run more smoothly), then this is your opportunity to ask questions. The presentation will begin with 5-min talks from five REU program coordinators. These coordinators run a wide range of programs and have experience ranging from 1 year to 12 years. They will mention things such as how many applicants their program receives, how they review those files, an outline of the non-research portion of their summer program, and what sorts of outcomes they collect so they can report them to NSF. Then, the coordinators will form a panel to answer questions. Shannon Bartelt-Hunt, Department of Civil Engineering Mark Griep, Department of Chemistry Don Becker, Redox Biolog

A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide election

The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an impor-tant reaction of extraordinary complexity. The DNA polymerase does not merely facilitate the insertion of dNMP, but it also performs rapid screening of substrates to ensure a high degree of fidelity. Several kinetic studies have determined rate constants and equilibrium constants for the elementary steps that make up the overall pathway. The information is used to develop a macro-scopic kinetic model, using an approach described by Ninio [Ninio J., 1987. Alternative to the steady-state method: derivation of reaction rates from first-passage times and pathway probabili-ties. Proc. Natl. Acad. Sci. U.S.A. 84, 663–667]. The principle idea of the Ninio approach is to track a single template/primer complex over time and to identify the expected behavior. The average time to insert a single nucleotide is a weighted sum of several terms, in-cluding the actual time to insert a nucleotide plus delays due to polymerase detachment from ei-ther the ternary (template-primer-polymerase) or quaternary (+nucleotide) complexes and time delays associated with the identification and ultimate rejection of an incorrect nucleotide from the binding site. The passage times of all events and their probability of occurrence are ex-pressed in terms of the rate constants of the elementary steps of the reaction pathway. The model accounts for variations in the average insertion time with different nucleotides as well as the in-fluence of G+C content of the sequence in the vicinity of the insertion site. Furthermore the model provides estimates of error frequencies. If nucleotide extension is recognized as a compe-tition between successful insertions and time delaying events, it can be described as a binomial process with a probability distribution. The distribution gives the probability to extend a primer/template complex with a certain number of base pairs and in general it maps annealed complexes into extension products

Kinetics of the DNA polymerase \u3ci\u3epyrococcus kodakaraensis\u3c/i\u3e

The polymerase chain reaction is one of the most important reactions in molecular biology. Single stranded DNA is copied in a complex series of steps, at the core of which lies the action of the DNA polymerase. At each nucleotide along the template, the polymerase screens the dNTP pool until it finds the complementary dNTP. The insertion of each dNMP is a balance between high fidelity and rapid elongation. In this study the kinetics of the β type polymerase pyrococcus kodakaraensis (KOD) is analyzed. The kinetics is influenced by reaction conditions such as the dNTP pool composition and temperature. In a previous study by Viljoen et al. [2005, A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection. Computational Biology and Chemistry 29, 101–110], a macroscopic kinetics expression of the polymerase chain reaction has been derived. The model contains four parameters that are intrinsic to a specific polymerase. The experiments to measure the temperature- dependence of the parameters for KOD DNA polymerase are reported. The results indicate that the optimal temperature for an equimolar dNTP pool is 72.5 °C and the optimum temperature shifts to lower temperatures when the dNTP pool composition is biased

Bacterial Protein Structures Reveal Phylum Dependent Divergence

Protein sequence space is vast compared to protein fold space. This raises important questions about how structures adapt to evolutionary changes in protein sequences. A growing trend is to regard protein fold space as a continuum rather than a series of discrete structures. From this perspective, homologous protein structures within the same functional classification should reveal a constant rate of structural drift relative to sequence changes. The clusters of orthologous groups (COG) classification system was used to annotate homologous bacterial protein structures in the Protein Data Bank (PDB). The structures and sequences of proteins within each COG were compared against each other to establish their relatedness. As expected, the analysis demonstrates a sharp structural divergence between the bacterial phyla Firmicutes and Proteobacteria. Additionally, each COG had a distinct sequence/structure relationship, indicating that different evolutionary pressures affect the degree of structural divergence. However, our analysis also shows the relative drift rate between sequence identity and structure divergence remains constant

PROFESS: a PROtein Function, Evolution, Structure and Sequence database

The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are ∼1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein–protein interaction networks

Förster Resonance Energy Transfer between Core/Shell Quantum Dots and Bacteriorhodopsin

An energy transfer relationship between core-shell CdSe/ZnS quantum dots (QDs) and the optical protein bacteriorhodopsin (bR) is shown, demonstrating a distance-dependent energy transfer with 88.2% and 51.1% of the QD energy being transferred to the bR monomer at separation distances of 3.5 nm and 8.5 nm, respectively. Fluorescence lifetime measurements isolate nonradiative energy transfer, other than optical absorptive mechanisms, with the effective QD excited state lifetime reducing from 18.0 ns to 13.3 ns with bR integration, demonstrating the Förster resonance energy transfer contributes to 26.1% of the transferred QD energy at the 3.5 nm separation distance. The established direct energy transfer mechanism holds the potential to enhance the bR spectral range and sensitivity of energies that the protein can utilize, increasing its subsequent photocurrent generation, a significant potential expansion of the applicability of bR in solar cell, biosensing, biocomputing, optoelectronic, and imaging technologies

Class-specific restrictions define primase interactions with DNA template and replicative helicase

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the β4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase–helicase interaction has co-evolved within each species

Primase structure and function

Primase is the ssDNA-dependent RNA polymerase that synthesizes RNA primers during DNA replication. In common with all DNA and RNA polymerases, primase has structural and functional features involved in polymer elongation. As RNA polymerase, it has structural and functional features for initiating chain synthesis. As a primase, it has structural and functional features for initiating chain synthesis on ssDNA. Using amino acid sequence analysis the structure of Escherichia coli primase responsible for binding zinc, at least three magnesium, and DnaB helicase has been identified. One of the magnesium binding motifs resembles the “active magnesium” motif found in all DNA and RNA polymerases. This motif can be considered to be involved in phosphodiester bond formation. The region with the putative zinc binding motif is the most highly conserved portion, including more than 25% of identical residues among bacterial primases. The function of the zinc finger may be to bind ssDNA in a sequence-specific manner. Primase has “RNAP” motif, a sequence found in all RNA polymerases which may be involved in chain initiation. Many of the observations concerning primer synthesis initiation in vivo have been reproduced by several of the in vitro assay systems. Important among these is that Okazaki fragments are initiated in vivo from d(CTG) most of the time. This trinucleotide initiation specificity has been shown to be an intrinsic property of pure primase in vitro. Using artificial ssDNA templates, primase has been shown to be the slowest and most error-prone polymerase yet studied. The rate-determining step is the first phosphodiester bond formed. Any protein which can influence either the dinucleotide synthesis rate or primase–ssDNA binding affinity will also play a key role in the regulation of primer synthesis initiation