Structural Biochemistry/Zinc fingers
A zinc finger is a self-contained domain that formed through the stabilization of a zinc ion binded to a pair of cysteines and a pair of histidines. In addition an inner hydrophobic attaches to it also. The discovery of the zinc finger revealed a new protein fold but also an important aspect of DNA recognition. In contrast to other DNA binding proteins that usually utilize the two-fold symmetry o the double helix, zinc finger are bonded together in a single file to recognize nucleic acid sequences of differing lengths. This standard design gives numerous combinations for the specific recognition of DNA or RNA. Therefore, it is expected that the zing finger is present in nature in great abundance. Also this explains why it is 3% of the genes in the human genome.
The zinc finger's most suitable role is for engineering proteins to find specific genes and target them. One application of them in 1994 used three finger protein to knock off the expression of oncogene transformed and modified into a mouse cell line. Also, targeting a slipped in zinc finger promoter led to the activation of a reporter gene. Therefore, genes can be switched on or off in a carefully chosen manner through the fusion of zinc finger peptides to repression or activation domains. In addition, it is possible that putting zinc fingers with the other effector domains like from nucleases or integrases, to make chimeric proteins, genomes could be controlled and modified. There are a couple applications of engineered zinc finger proteins, which can have therapeutic importance.
It has taken about ten years of research on the structure of chromatin to finally discover the nucleosome and a general depiction of its basic structure. Also it led to the discovery as the next level for the folding of DNA in the 300-A chromatin fiber. This led to an interest to what was labeled "active chromatin" back then. This is the chromatin that is involved in transcription or that was held in a balanced manner to do so, and finding a tractable system. This can lead to many possibilities of extracting relatively big quantities of material for biochemical and structural studies.
Robert Roeder and Donal Brown who studied the 5S RNA genes of Xenopus laevis, which transcribed by RNA polymerase III was interesting. They found out that the accurate starting stage of transcription needs the binding of a 40kDA protein factor, which is also known as factor A or transcription factor IIIA, which if extracted and purified from oocyte extracts. By utilizng a method called deletion mapping, it was found that this factor interacts with a region about 50 nucleotides long inside the gene, which is also known as the internal control region. The first eukaryotic transcription factor that was described was this.
Not very developed oocytes serve as place for 5s RNA molecules to be stored in in the form of 7S ribonucleoprotein particles, which each have a single 40-kDa protein, which was displayed to be identical with TFIIIa. TFIIIA hence, attaches to both the 5S RNA and its cognate DNA. In effect, it was thought that it may regulate the auto regulation the 5S gene transcription. Regardless of whether this autoregulation happens in vivo or not, the dual interaction gave an intriguing structural problem that could be gone about because of the big amounts of protein TFIIIA present in immature Xenopus oocytes.
A grad student named Miller, studied TFIIIA and found out an amazing repeating motif within the protein, which was later called a zinc finger because it had zinc and attached onto the DNA. This repeating structure was found through biochemistry and not through computer sequence analysis.
Preparation and Characterization of TFIIA
As Miller did his experiments over and over again for the published protocols for purifying the 7S RNP, he got very low yields, which were associated to dissociation. Brown and Roeder utilized buffers that had dithiothritol because the protein contained high level of cysteine content and EDTA to remove any contamination by metals, which hydrolyze nucleic acids. The gel filtration of the complex in 0.1 mM DTT led to a separate elution of protein and 5S RNA. However when the strong reducing agent sodium borohydride did not alter the complex, it was found out that the protein was not bound together by disulfide bridges and that a metal might have been associated with it. Then when after the particle was incubated with an array of chelating agents, the prevention of the particles breaking apart could occur with only the addition of Zn2+ beforehand and not by other types of metals. When analyzing and the partially purified 7S preparation solution using atomic absorption spectroscopy also showed that a good amount of concentration of Zn.
While these experiments occurred,Zinc was present in the 7S RNP at a ratio of two per particle. This was under the accurate value because their buffers had 0.5M or 1mM DTT, which possesses a high binding constant for Zn. Miller repeated this process with pure and intact particle preparations, without DTT. He concluded that the native 7S RNp has about 7 and 11 Zn ions.
↑ Klug Aaron(2009).The discovery of Zinc Fingers and their Applications. "Annual Review of Biochemistry", p. 3-6.