Decoding of the genetic information within the genomes of bacteria, plants and animals occurs in various steps that are sequentially linked together in space and in time within the cell. Spatiotemporal linkage or "coupling" is accomplished by specific proteins, which participate in multiple steps of the gene expression pathway through binding to the key informational molecules--DNA and messenger RNA (mRNA). This research seeks to understand coupling of the steps of gene expression through structural studies of the assembly of DNA-mRNA-protein complexes inside cells, using a novel blend of emerging nanotechnologies and molecular imaging methods. Magnetic nanoparticles will be used to recover specific molecular assemblies directly from cells. The composition of these assemblies will be probed with antibodies to specific proteins and with mass spectrometry. The structure of the assemblies will be studied by electron microscopy. Direct imaging of the gene assemblies will allow the investigators to evaluate proposed structural models of "molecular crosstalk" between the DNA and mRNA at specific locations along a gene as it is being decoded.
The last step in the protocol for the magnetic recovery of the nanoparticle-bound gene assemblies from cell extracts has yet to be perfected, making the project high risk, and development of efficient and effective recovery methods will be the first priority of the project. However, the risk is offset by the high potential payoff: the unique ability to recover gene complexes directly from cells at specific stages of the expression pathway. Knowledge of the spatiotemporal events of gene expression at the molecular level will be critical for the evaluation of models of gene networks that are emerging from various genome projects. A detailed understanding of genetic regulation is also essential for future development of biotechnology in various areas of national importance including agriculture, pharmaceuticals, ecology, computing, and energy production. Additionally, this project will provide training opportunities in the fields of nanotechnology and biomolecular imaging for undergraduate and graduate students from diverse ethnic and cultural backgrounds.
