Sandbox Reserved 1053: Difference between revisions

The Chromosome Determined Zinc Responsible (Czr) operon acts as described above, with Czr A acting as a regulator protein to the downstream gene Czr B. The Czr B gene codes for a Zn ²⁺ pump, so Czr A is responsible for controlling the transport of Zn ²⁺ out of the cell. Because of its role in regulating Zn ²⁺ levels, Czr A is considered a metal sensor protein. This allows Czr A to regulate the Czr operon to maintain an appropriate concentration of Zn ²⁺ inside the cell membrane.

18177-18182.</ref>. The Czr operon contains genes for the proteins Czr A and [http://proteopedia.org/wiki/index.php/3byr Czr B]. Czr B is a Zinc transport protein that exports Zn²⁺ out of the cell while Czr A regulates this process by controlling expression level of Czr B. When relatively low amounts of zinc are present in the cell Czr A will bind to the operator on the Czr operon, preventing the progression of RNA polymerase and thus inhibiting expression of Czr B. Decreased expression of Czr B results in a buildup of Zn²⁺ inside the cell, as there are fewer pumps to export Zn²⁺. Because Czr A and Czr B are transcribed as part of the same operon, an inhibitor of Czr A must be readily available to allow full transcription of Czr B when necessary. Czr A is allosterically inhibited by the binding of two Zn²⁺ ions, which is ideal in that this allows expression of Czr B to be dependent on the relative amount of Zn²⁺ in the cell. Czr A displays two different conformations; the first has a high affinity for DNA and has no Zn²⁺ ions bound to it (PDB code: 2KJB). In this conformation the <scene name='69/694220/A5_helices__dna_binding/2'>alpha 5 helices are aligned</scene>. Binding of zinc drives a conformational change (PDB code: 2KJC) in which the <scene name='69/694220/A5_helices_dna_binding/2'>alpha 5 helices become unaligned</scene>, changing the overall shape of the protein and significantly lowering its affinity for DNA (Figure 2). This allows for zinc transport to be self regulated. That is, when zinc concentration in the cell is high, zinc ions bind to Czr A, causing a conformational change which releases the bound DNA. DNA without Czr A bound is free to be transcribed and Czr B is again expressed, allowing for Zn²⁺ transport out of the cell. At low Zn²⁺ concentrations, Czr A represses RNA Polymerase activity, and Zn²⁺ ions are maintained inside the cell.

Ser 54, Ser 57, and His 58 are the primary sites of <scene name='69/694220/2kjb_colored/3'>DNA interaction</scene> in Czr A <ref name="critical"/>. These residues are likely to interact with the 5'-TGAA sequence found in the half-site of the DNA, where the alpha 4 helices (green) <scene name='69/694219/Czra_with_dna/2'>form an interaction with DNA</scene> (figure 3). Binding of two Zn ⁺² ions <scene name='69/694220/Dna_residues_when_inhibited/2'>pushes these residues out of their DNA binding conformation</scene>. Additionally, Val 42 and Gln 53 (lime green) are involved in the <scene name='69/694220/Val_42_and_gln_53/1'>DNA binding pocket</scene>. This conclusion was experimentally determined by mutagenesis of the Gln and Val residues with an Ala and measuring the mutant DNA binding capacity. The DNA bound state of Czr A was tested by using the known critical residues for DNA interactions <ref name="critical"/>. <scene name='69/694220/Dna_binding_residues/2'>Critical DNA binding residues</scene> Gln 53, Val 42 (aqua), Ser 54, Ser 57, and His 58 (lime) were individually mutated to Ala, and kinetic experiments were performed. Compared to wild type Czr A, mutating Gln53 and V42 residues resulted in an 11-fold and 160-fold decrease in K_a, respectively. Mutations to the main DNA interaction sites Ser 54, Ser 57, and His 58 result in binding similar to the inhibited non-DNA binding state, suggesting that these residues are essential to binding DNA. While the conformational change that occurs from the Zinc to DNA bound state of Czr A is small,the alpha 4 helices (shown in green in Figure 2) are slightly shifted. The loss of DNA binding in the mutagenesis experiements in combination with the lack of any other major physical changes between these two states further suggests that the alpha 4 helices are the location of DNA binding in Czr A. Experimental data can be found in table 1 from this same article.