Question

I asked my graduate student to reconstitute nucleosomes in vitro. She purified the 4 histones from E. coli, mixed them in equimolar amounts in a buffer with low salt to make octamers. She then tried to make nucleosomes by adding DNA to these octamers. Did she succeed in making the nucleosomes?

Answer 1

The answer to the question is most probably NO.

Before getting into that point we should know about how the DNA is packed within the cell. The DNA is a 2 m long molecule that is present in each and every cell. In order to make it possible the DNA is made into compact size with the help of proteins called Histone.

Histones are proteins that make the DNA into compact structure called Chromatid. When we observed through the microscope, we can see the string on a bead structure. The core histone proteins are H2A, H2B, H3, H4 and where H2A, and H2B exist as a dimer molecule and H3-H4 exists as a tetramer. They combined together to form the histone octamer. On a single bead, it covers 147 bp of DNA. The non-core histone proteins such as H1/H5 are called linker proteins, they help to connect the adjacent beads. Whereas the H1 proteins are required for the stability of histone octamer.

Nucleosomes are DNA with the histone proteins that observe like a single bead in the chromatid contains the core histone proteins, the DNA and the H1 protein.

Here only 4 histone proteins are isolated but it requires H1 proteins for the stability of the core histone octamer. For invitro nucleosome construction slow dilution from high salt concentration to low concentration is required and here she directly mixed in low salt concentration. Hence she will not suceed in making the nucleosome invitro.