Difference between revisions of "Part:BBa K346002"

Revision as of 06:43, 24 October 2010

PmerT promoter (mercury-responsive)

This part, PmerT, is a promoter from Tn21 mercury resistance (mer) operon. The mer operon of Tn21 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 structural gene – merPTAD. They are called merOP as a whole.

MerR, as a regulatory protein, always binds to merOP as a homodimer and enhances the occupancy of Ptpad by RNA polymerase regardless of the presence of Hg(II), although only after Hg(II)’s binding can the dimer stop preventing the formation of the open complex by RNA polymerase. Also, MerR repress its own expression independently of Hg(II)(Fig.1). For the characterization of merR, you may want to click the link here[1].

Fig.1. The model for the interaction of MerR and Hg(II) and its role in controlling PmerT transcription. Adapted from Jon L. Hobman, John Wilkie & Nigel L. Brown,2005. A: RNA polymerase (RNAP) transcribes merR from PmerR. MerR binds to the mer promoter/operator region (merOP) as a homodimer, recruits RNA polymerase, and represses transcription of merTPAD from PmerT. B: Hg(II) enters the bacterial cell by diffusion through the outer membrane, cytoplasm and inner membrane, and binds to three cysteine residues in the apo-MerR homodimer. The Hg-bound MerR homodimer causes an underwinding of merOP DNA, allowing RNAP to proceed with transcription of the resistance genes.

In our design, merR was isolated from the operon and assembled with constitutive promoters of certain strength to maintain its expression intensity at certain level. For the same reason, the divergent promoter Pr was also removed by deletion of its -35 region(Fig.2).

Fig.2. DNA sequence of the Tn21 mer operator 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. A: The divergently oriented promoters are marked by blue box and purple box, respectively. B: In our project, the expression intensity of MerR should be maintained exogenously, so the divergent promoter Pr (of MerR transcript) was also removed by deletion of its -35 region. The resulted promoter sequence is marked with a dark purple box Modified from (Hobman, Wilkie et al. 2005)

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Sequence and Features