Team:Groningen/Project/Promoters

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A promoter is a part of DNA involved in the regulation of gene transcription by RNA ploymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong.
A promoter is a part of DNA involved in the regulation of gene transcription by RNA ploymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong.

Revision as of 09:50, 25 September 2009

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A promoter is a part of DNA involved in the regulation of gene transcription by RNA ploymerase. In general RNA polymerase tends to bind weakly to a strand of DNA until a suitable promoter is encountered and the binding becomes strong.

In order to find different promoters to induce the different genes in the presence of different heavy metals we used the following list of databases and sites:

  1. KEGG
  2. NCBI
  3. Regtransbase

Copper Induced Promoters

Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. The toxicity of copper is largely due to its tendency to alternate between its cuprous, Cu(I), and cupric, Cu(II), oxidation states, differentiating copper from other trace metals, such as zinc or nickel. Under aerobic conditions, this redox cycling leads to the generation of highly reactive hydroxyl radicals that readily and efficiently damage biomolecules, such as DNA, proteins, and lipids.(needs a ref.). Most organisms have specialized mechanisms to deal with dangerous levels of heavy metals, like the production of efflux pumps. These genes are regulated by promoters, which are inducible by the respective metals.

E.coli

Promoter cusCp is associated with the two component system CusR and CusS for the copper induced transcription of genes involved in copper efflux (cusC, cusF, cusB and cusA, which is present on the genome of Escherichia coli str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following link. A second region, located at -53.5 from the transcription start site, is thought to bind CusR. Upon binding of CusR, the RNA polymerase is able to recognize the site and attach itself, and can also be found in the same link.

  • CusS, a sensory histidine kinase in a two-component regulatory system with CusR, is able to recognize copper ions, phosphorilate, and form a complex with CusR. It's a 480 amino acid long protein of which the sequence (aa and nt) can be found here allong with other information.
  • CusR, "Cu-sensing regulator", regulates genes related to the copper and silver efflux systems under anaerobic growth and under extreme copper stress in aerobic growth . It's a 227 amino acid long protein of which the sequence (aa and nt) can be found here along with other information.
Cu → CusS → +P → CusR → Activation of transription

The problem so far is the site of detection of copper. The CusS protein senses the external copper concentrations and not the internal. For our project it would be nice to have an internal sensor for the induction of the floatation genes, so it will float after uptake. In addition to CusR, three other systems involved in copper resistence are present (CueR, CpxR and YedW). All three have the same problem of sensing external copper instead of internal copper. The choice for CusR over CueR is based on the frequency of binding sites of both on the genome of E. coli (1 vs. 197 times), which gives CusR more chance of binding to our promoter.

Parts Registry

Promoter from the copper-sensitive CusR/CusS two component signal system in E.Coli (the CusR/CusS genes are not in parts registry, and are for external Cu concentration as mentioned before).

Abs: This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in E.coli. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of CusA, CusB, CusC and CusF genes coding the proteins of copper metabolic system. Was used by Saint-Petersburg Team of 2007 for constructing a copper biosensor system.

  • BBa_I760005
  • Cu-sensitive promoter
  • Part-only sequence (16 bp):
atgacaaaattgtcat

Other organisms

Mycobacterium tuberculosis
Abs.: Cu(I) binding to the CsoR–DNA complex induces a conformational change in the dimer that decreases its affinity for the DNA Liu2006.

Pseudomonas syringae
Abs.: The copper resistance (cop) operon promoter (Pcop) of Pseudomonas syringae is copper-inducible, and requires the regulatory genes copR and copS. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of copA Mills1994.

Sulfolobus solfataricus
Abs.: That CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the copMA gene cluster Ettema2006.

Lactococcus lactis
Abs.: Two regulatory genes (lcoR and lcoS) were identified from a plasmid-borne lactococcal copper resistance determinant and characterized by transcriptional fusion to the promoterless chloramphenicol acetyltransferase gene (cat). The transcription start site involved in copper induction was mapped by primer extension Khunajakr1999.

Zink Induced Promoters

Other organisms

Bacillus subtilis

Abs.: The Bacillus subtilis cation efflux pump czcD, which mediates resistance against Zn2+, Co2+, Ni2+ and Cu2+, is regulated by an ArsR-type repressor (CzrABS) as well Moore2005.


Streptococcus pneumoniae[6]