Bicyclomycin

Dr. William Widger has identified the target for the novel antibiotic, bicyclomycin, as the rho transcription termination factor in E. coli. Transcription termination by rho is a vital function in E. coli as well as other enteric gram-negative bacteria. Rho protein allows control of gene expression by regulating termination. Widger’s lab has shown that bicyclomycin acts as an anitermination factor that inhibits in vitro transcription termination. Bicyclomycin inhibits rho by interfering with protein-RNA interaction altering the 5' to 3' tracking of rho towards the RNA polymerase. The lab has putatively identified the antibiotic binding site on rho near the ATP hydrolysis pocket and further characterization is under investigation. Rho is a unique drug target which resembles the b-subunit of F1 ATPase. Currently, the lab is measuring drug binding and the effects of site mutations on bicyclomycin activity both in vitro and in vivo.

NADH:ubiquinone oxidoreductase (Complex I)

The first enzyme complex in the mammalian mitochondrial electron transport chain, NADH:ubiquinone oxidoreductase (NUO) is responsible for the vectorial transport of protons across the inner membrane coupled to electron transfer from NADH to ubiquinone. It contains at least 42 subunits and rivals the ribosome in protein complexity. NUO has been implicated in Parkinson's disease, Alzheimer's disease, and has been shown to be involved in the mitochondrial permeability pore transition initiating apoptosis. Mitochondriocytopathies have been estimated to occur in 1 per 10,000 live births and are most frequently found in NUO. Leigh syndrome, Leber's Hereditary Optic Neuropathy and neonatal lactic acidosis are caused by gene mutations in NUO. Stable semiubiquinone free radicals detected in isolated NUO are suggested to generate superoxide anions. Superoxide anions are implicated in aging and neuro-degenerative diseases. Widger’s lab is studying the production of reactive oxygen species (ROS) in isolated NUO using biochemical and biophysical techniques including electron paramagnetic resonance, enzyme kinetics and lipid reactivation. The production of ROS appears in part due to the mechanism of electron and proton transfer and aberrant enzymes increase ROS production. They are interested in how and why ROS production occurs and how this is linked to the enzymatic mechanism of NUO.

Photosynthesis

Cyanobacteria, once called blue-green alga, are prokaryotes which utilize photosynthesis as their main energy source. The photosynthetic apparatus found in cyanobacteria resembles closely to that found in higher plants. Widger’s lab has used Synechococcus PCC 7002 as a model organism to investigate the proteins involved in photosynthesis. PCC 7002 is a unicellular, naturally transformable species which can be manipulated by the use of molecular biological techniques to investigate the membrane associated multi-protein complexes found supporting and catalyzing light-energy transduction. The lab’s interest is in the structure/function studies of proteins involved in photosynthetic energy transduction. Light regulates gene expression in cyanobacteria and up regulates a set of specific transcripts in the dark. The mechanism of transcription regulation by light and transcript stability is also a research priority.

The physical genome map of PCC 7002 has been assembled from Not I, Sal I and Sfi I restriction fragments separated using pulsed-field gel electrophoresis. The genome size was shown to be 2.7 Mbp. A more detailed genome map is being created from overlapping cosmid clones using restriction fingerprinting and computer assisted contig assembly. This map is useful in the systematic identification of genes and eventual sequencing of the entire genome. This data will be used to monitor genome wide changes in transcription/translation in response to changes in carbon dioxide sequestration.