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Revision as of 12:08, 10 September 2009



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.

Different Promoters

In order to find different promoters to induce the different genes in the presence of different heavy metals I 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.


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):

Other organisms

Mycobacterium tuberculosis[2]

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

Pseudomonas syringae[3]

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.

Sulfolobus solfataricus[4]

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.

Lactococcus lactis[5]

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.

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 (Moore et al., 2005).

Streptococcus pneumoniae[6]

Abs.: Activation of the czcD promoter by SczA is shown to proceed by Zn2+-dependent binding of SczA to a conserved DNA motif. In the absence of Zn2+, SczA binds to a second site in the czcD promoter, thereby fully blocking czcD expression. A metalloregulatory protein belonging to the TetR family

Staphylococcus aureus

Abs.: In Staphylococcus aureus CzrA, a member of the ArsR/SmtB family of DNA binding proteins, functions as a repressor of the czr operon, that consists of czrA and the gene encoding the CzcD homologue CzrB (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999). CzrA-mediated repression is alleviated in the presence of Zn2+ and Co2+ (Xiong and Jayaswal, 1998; Kuroda et al., 1999; Singh et al., 1999).

Mercury Induced Promoters

Arsenicum Induced Promoters

Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including E. coli, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. (needs a ref.)


Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, 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 -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same link.

  • ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression.(KEGG)
  • ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in Escherichia coli has been used for removal of arsenite from contaminated water. (KEGG)

(ArsR)2-DNA --> ArsR-Ar + ArsR-Ar + DNA --> Activation of transription

The presence of all genes and promoters on the chromosome of E.coli makes the use of the arsRp for induction of the GVP cluster relatively straith forward. The promoter sequence of arsRp, with the upstream binding box for ArsR dimer, can either be synthesized completely with the required restriction sites, or acquired using PCR and carefully designed primers. It might even be an option to alter the -10/-35 promoter region for higher or lower transcription of the genes.

Other organisms

Bacillus subtilis[7]

In B. subtilis, an ArsR family repressor (ArsRBS) responds to As(III) and Sb(III) and regulates the ars operon encoding itself (ArsR), an arsenate reductase (ArsC), an arsenite efflux pump (ArsB) and a protein of unknown function (YqcK). The order in which ArsRBS recognises metals is as follows: As(III)>As(V)>Cd(II)~Ag(I).

A second protein, AseR, negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. The order in which AseR recognises metals is as follows: As(III)>As(V)


  • Need for specificity towards the wanted metals
  • Usable in E.coli

Planning and requirements:

  • Modelling
    • How do they react to metal, metal concentration vs expression of the protein after promotor (bv GFP)
  • Lab
    • Measuments
      • Expression of the gene behind the promotor (GFP). Relative to a constitutive promotor used with gvp.
    • Cu-promotor
    • Arc promotor