User:Marckemi

PBAD promoter
The  PBAD promoter is a part of the arabinose operon whose name derives from the genes it regulates transcription of: araB, araA, and araD. The  PBAD promoter is

adjacent to the PC promoter, which transcribes the araC gene in the opposite direction. araC encodes the AraC protein, which regulates activity of both the  PBAD and PC promoters. The cyclic AMP receptor protein CAP binds between the  PBAD and PC promoters, stimulating transcription of both when bound by cAMP.

Regulation of  PBAD

Transcription initiation at the  PBAD promoter occurs in the presence of high arabinose and low glucose concentrations. Upon arabinose binding to AraC, the N-terminal arm of AraC is released from its DNA binding domain via a “light switch” mechanism. This allows AraC to dimerize and bind the I1 and I2 operators. The AraC-arabinose dimer at this site contributes to activation of the PBADpromoter. Additionally, CAP binds to two CAP binding sites upstream of the I1 and I2 operators and helps activate the PBAD promoter. In the presence of both high arabinose and high glucose concentrations however, low cAMP levels prevent CAP from activating the PBAD promoter. It is hypothesized that PBAD promoter activation by CAP and AraC is mediated through contacts between the C-terminal domain of the α-subunit of RNA polymerase and the CAP and AraC proteins.

Without arabinose, and irregardless of glucose concentration, the PBAD and PCpromoters are repressed by AraC. The N-terminal arm of AraC interacts with its DNA binding domain, allowing two AraC proteins to bind to the O2 and I1 operator sites. The O2operator is situated within the araC  gene. An AraC dimer also binds to the O1 operator and represses the PC promoter via a negative autoregulatory feedback loop. The two bound AraC proteins dimerize and cause looping of the DNA. The looping prevents binding of CAP and RNA Polymerase, which normally activate the transcription of both PBAD and PC.

The spacing between the O2 and I1 operator sites is critical. Adding or removing 5 base pairs between the O2 and I1 operator sites abrogates AraC mediated repression of the PBAD promoter. The spacing requirement arises from the double helix nature of DNA, in which a complete turn of the helix is about 10.5 nucleotides. Therefore, adding or removing 5 base pairs between the O2 and I1 operator sites rotates the helix roughly 180 degrees. This reverses the direction that the O2operator faces when the DNA is looped and prevents dimerization of the O2 bound AraC with the bound I1 araC.