Question

As you may know, gene transcription begins with the assembly of the transcriptional machinery (TFIIA-H proteins) at the core promoter of genes. Where do you think core promoters are more likely to be located? Nucleosome DNA or Linker DNA?

Go ahead and formulate a hypothesis on the more likely location of core promoters of genes in DNA, and defend it with molecular biology arguments (in other words, why would one or the other be a preferred location for core promoters, based on what you know about nucleosome DNA and linker DNA?).

Then, I want you to imagine that you have access to a standard molecular genetics lab, and I want you to propose an experimental test of your hypothesis. You don’t need to get too technical, just a description of how you would design an experiment that tests your hypothesis, indicating samples, treatments, and the measurements or readouts that that you plan to take. Your imaginary lab has the following equipment:

1)  Antibodies for immunoprecipitation: these are molecules that can bind to several kinds of proteins, and allow you to physically pull them down (along with anything that is bound to them). In this case, imagine that you have access to antibodies that allow you to separate any proteins bound to DNA and isolate them along with any DNA bound to them!!

2)DNA sequencer: a machine that allows you to “read” the sequence of a given DNA sample.

3)Core promoter sequences: Information about what a typical core promoter sequence looks like.

So go ahead and design an experiment using any combination of one or more of the resources listed above, and do not forget to describe what results you expect to see, should your hypothesis be correct.

Your submission should be 200 words or fewer,

PLEASE use ONLY the equipment mentioned above.

Answer 1

Core promoter must be accessible by the transcription machinery, so that RNA polymerase II can transcribe the gene. Nucleosome, the building block of chromosome, consists of DNA wrap around the histone core protein, and each unit of nucleosome is connected to each other by the linker DNA. The tightly and closed wrapping of the histone core proteins, make the DNA sequence inaccessible to the proteins or enzymes that promote DNA replication, transcription or repair. Several histone modification (like acetylation on H2A or H3, the core histone protein) is reported which is essential to make the DNA sequence available for these enzymes. To promote transcription, core promoter might be located at the linker DNA. Literature search indicate the most probable location of the core promoter is the Nucleosome free region (NFR), which is a very long linker with approximately 140 bp in length and positioned just upstream of the transcription start site. Mostly the promoters (open) that transcribe constitutive genes harbor this NFR, while the promoters (covered) for the highly regulated gene contain nucleosome.

Flowchart of experiment

1. Cells are incubated with 37% paraformaldehyde to fix the transcription factor (TF)-DNA complexes
2. Cells are lysed with SDS-lysis buffer in the presence of protease inhibitor cocktail to prevent digestion of proteins by protease.
3. One half of these lysed cell suspension was sonicated by ultrasonication to break the crosslinked TF-DNA complex into shorter pieces with average of 100-150 bp in length.
4. Centrifuge the sample after sonication and discard the pellet which contains cell debris.
5. Other half the cell lysis suspension is digested by MNase (micrococcal nuclease), which digest the linker DNA
6. Supernatant was collected from both the cases, a) after sonication, and b) after MNase digestion
7. Incubate the supernatant with antibody specific to the transcription factor, whose promoter sequence has to be identified for prescribed time period.
8. Supernatant was discarded, and the precipitated product was washed with buffer containing lower to higher salt concentration
9. Finally the De-crosslinking agent (NaCl) was added to the mix, and DNA sample was collected, and send them for DNA sequencing.

Probable Result:

Core promoter binding site is on the nucleosome or nucleosome free region. can be detected by comparing the presence of its cognitive binding site in the samples extracted by sonication and MNase digestion. Core promoter DNA binding sequence might be detected in the sample that found after sonication, but any specific DNA binding sequence may not be present in the sample that extracted after MNase digestion, as this enzyme digest the linker DNA. Thus, the immunoprecipitation by antibody specific to the TF failed to find their cognitive partner in the reaction.