Question

4) FRET has been utilized to assess the mechanism by whic utilized to assess the mechanism by which RNA polymerase clears the promoter (3) AD Explain the process of FRET. What is needed to create a signal? How can this be used to measur distance/movement? (5 points) D ine authors determined that "DNA scrunching" was needed to create enough energy to clear the Promoter. Why does RNA polymerase need this extra boost to move away from the promoter and continue with elongation? (5 points)

Answer 1

A) FRET (Fluorescence resonance energy transfer) is a process of energy transferred from an excited donor fluorophore to acceptor fluorophore by intermolecular long-range dipole–dipole coupling. it is a distance-dependent physical energy tranfer technique. The excited donor chromophore emits light which is recieved by recipient chromophore of low energy. the recipient chromophore will be excited then and will emit flouroscence of different wavelength. Most commonly used flourophores are CFP(cyan flouroscent protein, emits green color) and YFP (yellow flouroscent protein, emitting yellow light).

When donor and acceptor are in close proximity i.e. 1–10 nm, the acceptor emission of flouroscent light will increase because of the intermolecular FRET. To measure the distance and affinity of two molecules, these molecules are labelled with different chromophore. On change of conformation or intensity of light emitted the change in distance will be measured. Farther the molecule will go, overlapping of light emitted will be reduced and there will be shift in emission area also and intensities will be reduced.

B) In DNA scrunching, RNA polymerase remains stationary and unwinds, pulls downstream DNA in transcription complex. Due to this pulling of DNA and passing of nucleotides through polymerase active site transcribes the DNA. The unwound DNA get accumulated in between the enzyme and hence called DNA "scrunching". This mechanism involves a lot of processes happening i.e. initially RNA polymerase re-winds and releases the downstream portion of the open DNA which ultimately releases the RNA into space. The RNA polymerase-promoter complex opens, causing elongation and opening for movement of complex.

In productive initiation, RNA polymerase re-winds, exposing upstream bases of the unwound DNA, ultimately breaking RNA polymerase and promoter interactions. The promoter get released and forms a transcription elongation complex.