As you may know, gene transcription begins with the assembly of the transcriptio

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.

Explanation / Answer

A promoter is a region from where the process of transcription for particular gene is initiated. These promoters are small DNA sequences up to 1000bp, upstream. The promoter region have attached with nucleosomes. Nucleosome is a structure in which DNA is bounded by 8 histone molecules and stabilizes the complex. Nucleosome is attaché with each other with a linker DNA. Promoter regulatory elements are located in nucleosome, upstream to transcription start site, is initiate the transcription process. This first evidence was derived from the experiment with S.cereviceae genome.

Core promoters are surrounded by the transcriptional start site (TSS) which contains binding site for RNA polymerase and it synthesizes RNA molecules. Our hypothesis for present experiment is that promoter regions are located towards the upstream of 5’ end with nucleosome.

To check our hypothesis DNA sequencer is the best technology to understand the sequences of gene. DNA sequencer read the correct sequence of DNA. Next generation DNA sequencing is the best platform available recently for sequencing,

First we extract DNA and amplify our gene of interest with PCR (design PCR primer in such way that is covers the promoter region). Amplified PCR products are now used as downstream application for NGS. Our sequence of interest will be read by the NGS machine and the data will be collected. Here we already have the core promoter element sequence information. Hence we can compare our NGS data with sequence we have, in a computational tool. If sequences are matched, our sequence of interest (promoter region) is present in our sample and it is accordance with our hypothesis. For further clarification we can also compare sequences of the gene of interest which will be transcribe. If it is matched and our promoter sequences are upstream to 5’ end of that gene, our hypothesis is correct.

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