Quantifying Gene Expression and Regulation in Living Cells by Fluorescence Fluctuation Imaging
Matthew L. Ferguson, Ph.D.
Department of Physics
Boise State University
Mechanisms of transcription and translation take the information encoded in the genome and make it "work" in cells, through the production of proteins defined by nucleic acid coding regions. This involves the coordination of many multi-subunit complexes about which most knowledge is inferred from ensemble and/or in vitro assays, giving a detailed but static picture. How these macromolecular machines coordinate in vivo remains unknown but recent advances in the application of fluctuation analysis to time resolved multi-color imaging can now give an unprecedented level of dynamic in vivo information. My talk will describe recent progress in this area and show examples from the regulation of the central carbon metabolism in bacteria and RNA transcription in humans. By Number and Brightness analysis, we quantify cell- to-cell variations in the expression of fluorescent proteins produced from two bacterial promoters controlling the switch between glycolysis and gluconeogenesis. This reveals strong transcriptional bursting and discriminates between two different molecular mechanisms of transcriptional repression. By two color, in vivo, RNA labeling, we visualize the rise and fall of the intron and exon during transcription of a single gene in human cells. By cross- correlation analysis, we determine the speed of transcription, co- transcriptional splicing and termination of the RNA transcript discerning correlation between elongation, splicing, cleavage and their relation to the chromatin environment.