Regulatory

Part:BBa_K346002

Designed by: Qianzhu Wu & Mei Chen   Group: iGEM10_Peking   (2010-10-12)

PmerT promoter (mercury-responsive)


This part, PmerT, is a promoter from Tn21 mercury resistance (mer) operon. The regulatory region of mer operon consists of two tightly overlapped, divergently oriented promoters – Pr and Ptpad.(Park, Wireman et al. 1992). Pr is the promoter of the regulatory protein gene, merR, and Ptpcad is for the transcription of the structure gene – merPTAD. Both of them are called merOP as a whole.


MerR-dimer.jpg

Fig.1. The mechanism of MerR mediated transcriptional activation. (A) The dimeric MerR regulator binds to the operator region of the promoter and recruits RNA polymerase, forming a ternary complex. Transcription is slightly repressed because the apo-MerR regulator dimer has bent the promoter DNA such that RNA polymerase does not contact it properly. (B) Upon binding the cognate metal ions (shown as cyan circles) the metallated MerR homodimer causes a realignment of the promoter such that RNA polymerase contacts the -35 and -10 sequences leading to open complex formation and transcription. Adapted from Brown et al.


The key sequence for MerR’s binding is a region of interrupted dyad symmetry (19bp) located between the -35 and -10 haxamers of Ptpcad (The top strand). And the structure of Pr (botton strand) is similar to Ptpcad in a divergent orientation. The -10 hexamers of Ptpcad and Pr actually overlap by four bases. When the apo-MerR dimer bind to the dyad symmetrical operator DNA between the -35 and – 10 elements of mercury inducible promoter, PmerT, which has a unusually long spacer of 19 bp for MerR to bind on, the binding of RNA polymerase is inhibited(Fig.2). The Hg-bound MerR can result in an a structural distortion of PmerT, allowing the RNA polymerase contacts to be made, leading to the expression of downstream genes. This model of transcription activation indicates that the apo-MerR and Hg-bound MerR have a competing relationship. The threshold of PmerT is controlled by the expression level of MerR. As a consequence, the sensitivity of Hg(II) in cell can be manipulated.

PmerT111.jpg

Fig.2. DNA sequence of the Tn21 mer operon promoter region. The MerR binding site on PmerT is marked by a box. The -35 and -10 regions for both PmerR and PmerTPAD are marked with boxes, and the dyad symmetrical DNA sequence that MerR recognizes and binds to is marked with arrows under the DNA sequence. The divergently oriented promoters are marked by blue box and purple box, respectively.


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Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//function/sensor/metal
//promoter
//regulation
Parameters
chassisit works in DH5 alpha
n/aPmerT promoter (mercury-responsive)