PBAD promoter

P_BAD (systematically araBp) is a promoter found in bacteria and especially as part of plasmids used in laboratory studies. The promoter is a part of the arabinose operon whose name derives from the genes it regulates of: araB, araA, and araD. In E. coli, the P_BAD promoter is adjacent to the P_C promoter (systematically araCp), which transcribes the araC gene in the opposite direction. araC encodes the AraC protein, which regulates activity of both the P_BAD and P_C promoters. The cyclic AMP receptor protein CAP binds between the P_BAD and P_C promoters, stimulating transcription of both when bound by cAMP.

Regulation of P_BAD

Transcription initiation at the P_BAD promoter occurs in the presence of high L-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 P_BADpromoter. Additionally, CAP binds to two CAP binding sites upstream of the I₁ and I₂ operators and helps activate the P_BAD promoter. In the presence of both high arabinose and high glucose concentrations however, low cAMP levels prevent CAP from activating the P_BAD promoter. It is hypothesized that P_BAD 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 regardless of glucose concentration, the P_BAD and P_Cpromoters 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 P_C 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 P_BAD and P_C.

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 P_BAD 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.

The P_BAD promoter on expression plasmids

<br /> The P_BAD promoter allows for tight regulation and control of a target gene in vivo. As explained above, P_BAD is regulated by the addition and absence of arabinose. As tested, the promoter can be further repressed with reduced levels of cAMP through the addition of glucose. Plasmid vectors have been constructed and tested with a selectable marker (Cm^R in this case), origin of replication, araC and operons, multiple cloning site and P_BAD promoter. Studies show that vectors are highly expressed and can be used, in combination with chromosomal null alleles, to study loss of function of essential genes.

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