b. Shown below is part of Figure 3 of the paper by Ferreira et al. 2007 (Molecul
ID: 192621 • Letter: B
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b. Shown below is part of Figure 3 of the paper by Ferreira et al. 2007 (Molecular and Cellular Biology, 27:4037-4048). In this paper the authors assemble nucleosomes from a 181 bp piece of DNA with the Cy3 fluorochrome at one end and the Cy5 fluorochrome at the other end, and added histones. They use all unmutated (wild type H2A, H2B, H3 and H4) histones, or substitute mutated H3 or mutated H4 histones for the wild type equivalents. The mutated histones have the amino terminal tails deleted. The figure below uses FRET to study the nucleosomes assembled with either H3 or H4 in which the amino terminal tail has been deleted. Fluorescence resonance energy transfer (FRET) is a technique where the Cy3 fluorochrome, when excited by the appropriate wavelength (lightning bolt in Figure 3A), can transfer energy to the Cy5 fluorochrome, causing it to fluoresce. The energy transfer is distance dependent, so the amount of fluorescence emitted by Cy5 is inversely proportional to its distance from the Cy3 fluorochrome. FerreiraFig3ps1 Figure 3: Deletion of the H3 tail affects DNA end-to-end FRET. (A) Nucleosomes were assembled onto DNA containing Cy3 on one end and Cy5 on the other. Once assembled, the two arms of DNA were brought into close enough proximity to generate a FRET signal. (B) Full-length refers to nucleosomes assembled from wild type (WT) histones; gH3 refers to nucleosomes assembled with H3 histones lacking the amino terminal tail; gH4 refers to nucleosomes assembled with H4 histones lacking the amino terminal tail. What does Figure 3B tell you about the effect of deleting the amino terminal tail of histone H3? What does Figure 3B tell you about the effect of deleting the amino terminal tail of histone H4? Which deletion has a larger effect on the nucleosome structure? How did you decide this? What is the difference in the structure of the nucleosomes made with all wild type histones and the nucleosomes made with the H3 mutant histones?
Figure 3: Deletion of the H3 tail affects DNA end-to-end FRET. (A) Nucleosomes were assembled onto DNA containing Cy3 on one end and Cy5 on the other. Once assembled, the two arms of DNA were brought into close enough proximity to generate a FRET signal. (B) Full-length refers to nucleosomes assembled from wild type (WT) histones; gH3 refers to nucleosomes assembled with H3 histones lacking the amino terminal tail; gH4 refers to nucleosomes assembled with H4 histones lacking the amino terminal tail.
Be specific but explain it in a way that is easy to understand.
What does Figure 3B tell you about the effect of deleting the amino terminal tail of histone H3?
What does Figure 3B tell you about the effect of deleting the amino terminal tail of histone H4?
Which deletion has a larger effect on the nucleosome structure? How did you decide this?
What is the difference in the structure of the nucleosomes made with all wild type histones and the nucleosomes made with the H3 mutant histones?
181bp 601.2 ONA fragment Chromatin assembly Site exposure or breathing Cy5 The dyes are well sepa rated hence low FRET DNA ends are brought into close proximity and can undergo FRET DNA ends move apart and FRET is reduced. 100 Full-length 9H4 9H3 NaCI concentration (mM)Explanation / Answer
Figure 3B gives us information about effect of deleting the amino acid terminal tail of Histone H3. It effects the FRET to FRET signaling. According to the figure, it is observed that when mutated H3 is present, there is absence of the amino terminal tail in the histone H3 protein. It is observed that in such case the FRET signal is very less and it is not able to transmit the signal from Cyt3 to Cyt5 appropriately. Since, the amino acid is lacking, the signal is disrupted keeping FRET level low.
In figure 3B, there is effect of deleting amino terminal tail of histone H4 also observed where the protein lacks the amino acid signal. It is observed that if mutated H4 is used, there is FRET signaling present but the level is not at its extreme high. There is average amount of signaling done for the same.
The deletion of the WT (wild type) histone has large effect on the nucleosome as assembling is not done properly. Due to the absence of the wild – type, the FRET signaling becomes comparatively weak that poses a threat. It is decided as per the graph where it is clearly indicated that full length nucleosome with wild type of histone gives high degree of FRET signaling.
There is difference in the structure made from all wild type histones and nucleosome made from H3 mutant histones. The assembly process is stronger when wild types are used as the cytochrome on both sides tends to send impulse and there is energy transmission from cyt3 to cyt5 which helps in bringing DNA ends to close proximity and hence assists with better compact nucleosome structure formation. When H3 mutated version is used, it lack amino terminal which creates bad signaling and prevents the molecules from coming close. The FRET signaling observed is also low.
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