PBAD promoter

{{technical|date=July 2014}}

thumb|'''Figure 1. Expression of ''araB'', ''araA'' and ''araC'' in the presence of arabinose.''' In the presence of L-arabinose, arabinose binds to the arabinose binding pocket sites of AraC, causing AraC to dimerize at the I₁ and I₂ operators. This allows access for CAP to bind to the CAP-binding sites, which in turn helps recruit RNA Polymerase to both P_BAD and P_C promoters and activates transcription.|415x415px

'''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 transcription of: ''araB'', ''araA'', and ''araD''.<ref name=":0">Schleif R. AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev (2010) 1–18.</ref><ref name=":1">Schleif R. AraC protein: a love-hate relationship. Bioessays 2003, 25:274-282.</ref> 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.<ref name=":2">Reed WL, Schleif RF. Hemiplegic Mutations in AraC Protein. J. Mol. Biol. (1999) 294, 417-425.</ref><ref name=":3">Lobell RB, Schleif RF. DNA looping and unlooping by AraC protein. Science. 1990 Oct 26;250(4980):528-32.</ref><ref>Soisson SM, MacDougall-Shackleton B, Schleif R, Wolberger C. Structural basis for ligand-regulated oligomerization of AraC. Science. 1997 Apr 18;276(5311):421-5.</ref> The cyclic AMP receptor protein CAP binds between the P_BAD and P_C promoters, stimulating transcription of both when bound by cAMP.<ref name=":4">Dunn T.M., Schleif R. Deletion Analysis of the Escherichia coli ara PC and PBAD Promoters. J.Mol. Biol. (1984) 180, 201-204.</ref>

==Regulation of P_BAD==

Transcription initiation at the P_BAD promoter occurs in the presence of high L-arabinose and low glucose concentrations.<ref name=":5">Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. Jul 1995; 177(14): 4121–4130.</ref> Upon arabinose binding to AraC, the N-terminal arm of AraC is released from its DNA binding domain via a “light switch” mechanism.<ref name=":0" /><ref name=":1" /> This allows AraC to dimerize and bind the I₁ and I₂ operators.<ref name=":2" /> The AraC-arabinose dimer at this site contributes to activation of the P_BADpromoter.<ref name=":1" /> Additionally, CAP binds to two CAP binding sites upstream of the I₁ and I₂ operators and helps activate the P_BAD promoter.<ref name=":4" /> In the presence of both high arabinose and high glucose concentrations however, low cAMP levels prevent CAP from activating the P_BAD promoter.<ref name=":5" /> 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.<ref>Johnson CM, Schleif RF. Cooperative Action of the Catabolite Activator Protein and AraC In Vitro at the araFGH Promoter. J Bacteriol. Apr 2000; 182(7).</ref>

thumb|'''Figure 2. Expression of ''araB'', ''araA'' and ''araC'' does not occur when arabinose is not present.''' In the absence of arabinose, AraC dimerizes while bound to the O₂ and I₁ operator sites, looping the DNA. The looping prevents binding of CAP and RNA Polymerase, which normally activate the transcription of both P_BAD and P_C.|416x416px

Without arabinose, and regardless of glucose concentration, the P_BAD and P_Cpromoters are repressed by AraC.<ref name=":1" /><ref name=":5" /> 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.<ref name=":0" /> 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.<ref name=":1" /> The two bound AraC proteins dimerize and cause looping of the DNA.<ref name=":2" /><ref name=":3" /> The looping prevents binding of CAP and RNA Polymerase, which normally activate the transcription of both P_BAD and P_C.

{|class="wikitable"

| Transcription by P_BAD

| '''High Arabinose'''

| '''Low Arabinose'''

|- | '''High Glucose'''

| Repressed

| Repressed

|- | '''Low Glucose'''

| Active

| Repressed

|}

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.<ref name=":0" /> 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''.<ref name=":0" /><ref name=":1" />

==The P_BAD promoter on expression plasmids==

thumb|'''Figure 3. A representation of the PBAD 33 promoter on a plasmid with common cis-acting regions.''' Abbreviations are defined as the phagemid origin (f1 origin), chloramphenicol resistance (CmR), plasmid origin (p15A ori), araC gene (araC), araC operator sites (araC O2 and O1), CAP-binding site (CAP BS), araC inducer sites (I1/I2), PBAD promoter (pBAD) and the multiple cloning site (MCS).|375x375px <br /> The P_BAD promoter allows for tight regulation and control of a target gene ''in vivo''.<ref name=:5 /> 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.<ref name=:5 /> 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.<ref name=:5 />

==References== {{Reflist}}

Category:Gene expression