A Study of Transcriptional Activation by the Transcription Factor Gal4 in Saccharomyces cerevisiae by 3D Orbital Tracking and in vivo RNA labelling
Additional Funding Sources
The project described was supported by NIH grant Southwest Idaho Bridges to the Baccalaureate Award No. R25GM123927, additional support provided by NIH Grant Nos. P20GM103408 and P20GM10909, and the Boise State University Biomolecular Research Center, and Research Corporation for Science Advancement and the Gordon and Betty Moore Foundation through Grant GBMF5263.10, and by National Institute of General Medical Sciences through Grant 1R15GM123446.
Abstract
Understanding what is going on at the molecular level within individual cells is challenging. But deciphering stochastic biomolecular processes is crucial for our understanding of gene transcription and the intricacies of cellular metabolism. With 3D orbital tracking, we are able to visualize and monitor pre-mRNA and transcription factors in real-time using fluorescent tagging within yeast cells at a high sampling rate. Our study demonstrates that we can track these molecules of interest, fluorescent-labeled GFP (pre-mRNA) and JF 646 dye (transcription factors), during the process of transcribing a gene that codes for metabolizing galactose. This method allows us to directly observe the movement of a single molecule and determine the time lag between the GAL4 transcription factor binding to DNA and the activation of the mRNA synthesis. The data we collected improves our knowledge of the details of transcriptional kinetics and how single celled eukaryotic organisms regulate transcription. This will expand our research on the transcription processes in similar genes and in multicellular organisms like humans. 3D orbital tracking opens up a new window for exploring fundamental biochemical processes through a dynamic view of single fluorescent molecules in living systems at high speed.
A Study of Transcriptional Activation by the Transcription Factor Gal4 in Saccharomyces cerevisiae by 3D Orbital Tracking and in vivo RNA labelling
Understanding what is going on at the molecular level within individual cells is challenging. But deciphering stochastic biomolecular processes is crucial for our understanding of gene transcription and the intricacies of cellular metabolism. With 3D orbital tracking, we are able to visualize and monitor pre-mRNA and transcription factors in real-time using fluorescent tagging within yeast cells at a high sampling rate. Our study demonstrates that we can track these molecules of interest, fluorescent-labeled GFP (pre-mRNA) and JF 646 dye (transcription factors), during the process of transcribing a gene that codes for metabolizing galactose. This method allows us to directly observe the movement of a single molecule and determine the time lag between the GAL4 transcription factor binding to DNA and the activation of the mRNA synthesis. The data we collected improves our knowledge of the details of transcriptional kinetics and how single celled eukaryotic organisms regulate transcription. This will expand our research on the transcription processes in similar genes and in multicellular organisms like humans. 3D orbital tracking opens up a new window for exploring fundamental biochemical processes through a dynamic view of single fluorescent molecules in living systems at high speed.
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