Uncovering Coordination of Gene Copying and Reading in Live Cells
Summary
A live-cell imaging technique was devised in this study to track the movement of replisomes and transcription dynamics during transcription-replication contacts in real time. The findings suggest that a wave of partial transcriptional suppression occurs prior to the replication fork, which may improve fork advancement across the transcribed gene. Furthermore, real-time detection of replication and transcription conflicts demonstrated that they had a negative influence on both processes, resulting in fork stalling or slowing as well as lower transcription levels during gene replication. These impacts were found to have various trade-offs in different cell subpopulations.
Research Criteria
The criteria for the research involved looking at the relationship between transcription, which is the process of converting DNA into RNA, and replication, which is the process of copying DNA. The researchers wanted to understand how these two processes are coordinated within cells, as they both take place in the same region of the cell and use the same set of enzymes. To investigate this, the researchers used a technique called dual-color live-cell imaging, which allows them to visualize both transcription and replication simultaneously in individual cells. They then analyzed the data to see how the two processes were coordinated and how they were affected by changes in the environment, such as changes in the concentration of nutrients or the presence of stressors.
Sample Type
Yeast; Single live cells
Result—Simultaneous Monitoring of Replisome Progression and Transcription Dynamics at an Inducible TRC
Their methodology for the observation of an inducible conflict between a singular replisome, which moves unidirectionally, and a gene undergoing intensive transcription relies on an amalgamation of established techniques that measure the progression of replisomes and the dynamic nature of transcription through live-cell imaging. Real-time monitoring of replication is achieved by arrays of bacterial operator sequencers, which are bound by a cognate repressor linked with a fluorescent protein, and permit visualization of specific loci of the chromosome as fluorescent dots. The moment when each of these arrays undergoes replication can be tracked, as the duplication of the array results in the additional recruitment of fluorescently labeled repressor protein, causing an upsurge in the intensities of the fluorescent dots.
Fig.1 Real-time monitoring of replisome progression and transcription dynamics at the same locus in single live cells. (Tsirkas, 2022)
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Reference
- Tsirkas, I.; et al. Transcription-replication coordination revealed in single live cells. Nucleic Acids Research. 2022, 50(4): 2143-2156.
