Lymphangiogenesis – pt3 Flashcards
Q1: Why is gene transcription described as discontinuous?
A: Gene expression occurs in bursts, not as a continuous process. These bursts vary in frequency and intensity, depending on regulatory inputs.
Q2: What determines the specificity of transcription factor (TF) binding to DNA?
A: TFs randomly scan the genome and bind short-lived to many sites. High-affinity binding sites retain TFs longer, allowing regulation of transcription.
Q3: What does molecular imaging reveal about TF activity?
A: It shows real-time movement, binding duration (dwell time), and binding frequency, helping us understand how TFs control transcription dynamically.
Q4: What is the difference between specific and non-specific TF binding?
A: Specific binding involves high-affinity target sequences (longer dwell times); non-specific binding is low-affinity scanning (shorter dwell times).
Q5: How does transcription factor behavior scale up to tissue function?
A: TF activity affects gene expression → which drives cell behavior → influencing tissue morphogenesis (e.g. vascular development).
Q6: What is the Rolex system used for?
A: Real-time imaging of mRNA production at the gene locus using MS2 loops and fluorescent chaperones.
Q7: What are MS2 loops?
A: RNA structures engineered into mRNA that can be bound by labeled proteins (e.g., MCP-RFP) for visualization.
Q8: What does single molecule tracking (SMT) measure?
A: The movement, binding frequency, and dwell time of individual TFs in live cells.
Q9: What are the two main TF states revealed by SMT?
A: Bound state (attached to chromatin) and diffusing state (searching for targets).
Q10: What is the average dwell time for specific TF-DNA binding?
A: ~5 seconds (long-lived). Non-specific dwell time is ~0.5 seconds.
Q11: What is HaloTag?
A: A modified enzyme fused to TFs for fluorescent labeling with high specificity and low background.
Q12: What type of microscopy is used for HaloTag tracking?
A: A form of live-cell fluorescence microscopy, tuned to visualize protein activity in the nucleus.
Q14: What does a TF trajectory map show?
A: The path of individual proteins moving within the nucleus and interacting with chromatin.
Q15: What are the scales of biological investigation discussed?
A: Molecular (TF binding), Cellular (cell behavior), and Tissue (vessel formation).
Q16: If a TF shows short dwell times only, what can you infer?
A: It is mainly scanning the genome with low affinity—not binding specific regulatory elements.
Q17: How does MS2-GFP and gene locus-GFP help in mRNA imaging?
A: Overlapping red (mRNA) and green (gene locus) signals confirm transcription is occurring at that locus.
Q18: What would indicate a gene is a fast, high-frequency burster?
A: Many short intervals between large mRNA production spikes during live imaging.
Q19: Why is labeling both RNA and DNA loci necessary in Rolex?
A: To confirm that detected mRNA is being transcribed from the observed gene locus, not elsewhere.
Q20: How does combining mRNA imaging and TF tracking enhance understanding?
A: It allows correlation of TF behavior (binding/diffusion) with actual transcription output at single-cell level.
