As microscopy tools get better and better, scientists have been able to visualize structures on a smaller and smaller scale. Researchers have now been able to capture an enzyme called RNA polymerase as it carries out one of life's most essential processes: the copying of a DNA sequence into an RNA molecule that can be used by the cell to make protein, for example. For the first time, researchers have seen the process of transcription as it starts, at the level of an individual gene. The findings have been reported in Nature Communications.
"This is the first time someone has looked at RNA polymerase phosphorylation dynamics in a single-copy gene," said study leader and postdoctoral researcher Linda Forero-Quintero Forero, Ph.D. of Colorado State University.
At one time, researchers studying transcription used models called gene arrays, in which hundreds of copies of a gene could be transcribed; but that's not how the process naturally happens. In this work, the researchers exposed cells growing in culture to fluorescent antibodies that would illuminate various parts of the transcription mechanism. The cell line contains a reporter gene that lights up when transcribed. They were able to see the transcription enzyme RNA polymerase II (RNAP2) as some of the amino acids making up the enzyme gain phosphate groups (and are phosphorylated). Three steps in the transcription cycle were highlighted by different colors, and the intensity of the fluorescence also changed.
The scientists were able to use this data to model the spatiotemporal movement of RNAP2 throughout a transcription cycle.
"The interdisciplinary science here is a fantastic blending of new experimental capabilities and a new approach for mechanistic computational modeling of single-cell dynamics, both of which are very novel in their respective fields," noted study supervisor Brian Munsky, an associate professor in chemical and biological engineering at CSU.
The research team was also able to create a model based on their results, and use it to learn more about transcription. For example, they've suggested that for every burst of transcriptional activity, a cluster of five to forty RNA polymerases gather around a gene's promoter. Eventually, about 46 percent of that group succeeds in transcribing a copy of a gene. They also found that each RNA took about five minutes before being transcribed and released, although I assume that figure may be different for different genes depending on their size and other features.
The scientists are hopeful that this technology has other applications, and could be combined with CRISPR to investigate individual genes under manipulation. One day soon, it may be possible to actually see dysfunctional processes, as they occur in disease, while they happen at the level of an individual cell or single gene.
"The ability to resolve the spatial and temporal dynamics of the transcription cycle, in one gene, is the most exciting aspect of this work," Forero said.