PBAD promoter

Last updated
Figure 1. Expression of araB, araA and araC in the presence of arabinose. In the presence of 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 PBAD and PC promoters and activates transcription. Figure 2. Expression of araB, araA and araC in the presence of arabinose..png
Figure 1. Expression of araB, araA and araC in the presence of arabinose. In the presence of 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 PBAD and PC promoters and activates transcription.

PBAD (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. [1] [2] In E. coli , the PBAD promoter is adjacent to the PC promoter (systematically araCp), which transcribes the araC gene in the opposite direction. araC encodes the AraC protein, which regulates activity of both the PBAD and PC promoters. [3] [4] [5] The cyclic AMP receptor protein CAP binds between the PBAD and PC promoters, stimulating transcription of both when bound by cAMP. [6]

Promoter (genetics) a region of DNA that initiates transcription of a particular gene

In genetics, a promoter is a region of DNA that leads to initiation of transcription of a particular gene. Promoters are located near the transcription start sites of genes, upstream on the DNA . Promoters can be about 100–1000 base pairs long.

Bacteria A domain of prokaryotes – single celled organisms without a nucleus

Bacteria are a type of biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs, radioactive waste, and the deep biosphere of the earth's crust. Bacteria also live in symbiotic and parasitic relationships with plants and animals. Most bacteria have not been characterised, and only about 27 percent of the bacterial phyla have species that can be grown in the laboratory . The study of bacteria is known as bacteriology, a branch of microbiology.

Plasmid small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently

A plasmid is a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism, such as by providing antibiotic resistance. While the chromosomes are big and contain all the essential genetic information for living under normal conditions, plasmids usually are very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation.

Contents

Regulation of PBAD

Transcription initiation at the PBAD promoter occurs in the presence of high arabinose and low glucose concentrations. [7] Upon arabinose binding to AraC, the N-terminal arm of AraC is released from its DNA binding domain via a “light switch” mechanism. [1] [2] This allows AraC to dimerize and bind the I1 and I2 operators. [3] The AraC-arabinose dimer at this site contributes to activation of the PBAD promoter. [2] Additionally, CAP binds to two CAP binding sites upstream of the I1 and I2 operators and helps activate the PBAD promoter. [6] In the presence of both high arabinose and high glucose concentrations however, low cAMP levels prevent CAP from activating the PBAD promoter. [7] 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. [8]

Glucose A simple form of sugar

Glucose is a simple sugar with the molecular formula C6H12O6. Glucose is the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using energy from sunlight. There it is used to make cellulose in cell walls, which is the most abundant carbohydrate. In energy metabolism, glucose is the most important source of energy in all organisms. Glucose for metabolism is partially stored as a polymer, in plants mainly as starch and amylopectin and in animals as glycogen. Glucose circulates in the blood of animals as blood sugar. The naturally occurring form of glucose is d-glucose, while l-glucose is produced synthetically in comparatively small amounts and is of lesser importance. Glucose is a monosaccharide containing six carbon atoms and an aldehyde group and is therefore referred to as an aldohexose. The glucose molecule can exist in an open-chain (acyclic) and ring (cyclic) form, the latter being the result of an intramolecular reaction between the aldehyde C atom and the C-5 hydroxyl group to form an intramolecular hemiacetal. In water solution both forms are in equilibrium and at pH 7 the cyclic one is the predominant. Glucose is a primary source of energy for living organisms. It is naturally occurring and is found in fruits and other parts of plants in its free state. In animals glucose arises from the breakdown of glycogen in a process known as glycogenolysis.

Activation in (bio-)chemical sciences generally refers to the process whereby something is prepared or excited for a subsequent reaction.

In molecular biology and genetics, upstream and downstream both refer to relative positions of genetic code in DNA or RNA. Each strand of DNA or RNA has a 5' end and a 3' end, so named for the carbon position on the deoxyribose ring. By convention, upstream and downstream relate to the 5' to 3' direction respectively in which RNA transcription takes place. Upstream is toward the 5' end of the RNA molecule and downstream is toward the 3' end. When considering double-stranded DNA, upstream is toward the 5' end of the coding strand for the gene in question and downstream is toward the 3' end. Due to the anti-parallel nature of DNA, this means the 3' end of the template strand is upstream of the gene and the 5' end is downstream.

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 PBAD and PC. Ara abs.png
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 PBAD and PC.

Without arabinose, and regardless of glucose concentration, the PBAD and PC promoters are repressed by AraC. [2] [7] 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. [1] The O2 operator 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. [2] The two bound AraC proteins dimerize and cause looping of the DNA. [3] [4] The looping prevents binding of CAP and RNA Polymerase, which normally activate the transcription of both PBAD and PC.

Repressor DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers

In molecular genetics, a repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to the mRNA and prevents translation of the mRNA into protein. This blocking of expression is called repression.

Negative feedback occurs when some function of the output of a system, process, mechanism is fed back in a manner that tends to reduce the fluctuations in the output, whether caused by changes in the input or by other disturbances

Negative feedback occurs when some function of the output of a system, process, or mechanism is fed back in a manner that tends to reduce the fluctuations in the output, whether caused by changes in the input or by other disturbances.

Autoregulation is a process within many biological systems, resulting from an internal adaptive mechanism that works to adjust that system's response to stimuli. While most systems of the body show some degree of autoregulation, it is most clearly observed in the kidney, the heart, and the brain. Perfusion of these organs is essential for life, and through autoregulation the body can divert blood where it is most needed.

Transcription by PBAD

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 PBAD promoter. [1] 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 O2 operator faces when the DNA is looped and prevents dimerization of the O2 bound AraC with the bound I1araC. [1] [2]

Base pair unit consisting of two nucleobases bound to each other by hydrogen bonds: either adenine–thymine or guanine–cytosine in natural DNA (additional types occur in RNA)

A base pair (bp) is a unit consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA and RNA. Dictated by specific hydrogen bonding patterns, Watson–Crick base pairs allow the DNA helix to maintain a regular helical structure that is subtly dependent on its nucleotide sequence. The complementary nature of this based-paired structure provides a redundant copy of the genetic information encoded within each strand of DNA. The regular structure and data redundancy provided by the DNA double helix make DNA well suited to the storage of genetic information, while base-pairing between DNA and incoming nucleotides provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase transcribes DNA into RNA. Many DNA-binding proteins can recognize specific base-pairing patterns that identify particular regulatory regions of genes.

DNA Molecule that encodes the genetic instructions used in the development and functioning of all known organisms and many viruses

Deoxyribonucleic acid is a molecule composed of two chains that coil around each other to form a double helix carrying genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids; alongside proteins, lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life.

Nucleotide biological molecules that form the building blocks of nucleic acids

Nucleotides are molecules consisting of a nucleoside and a phosphate group. They are the basic building blocks of DNA and RNA.

The PBAD promoter on expression plasmids

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). PBAD33.png
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).


The PBAD promoter allows for tight regulation and control of a target gene in vivo. [7] As explained above, PBAD 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. [7] Plasmid vectors have been constructed and tested with a selectable marker (CmR in this case), origin of replication, araC and operons, multiple cloning site and PBAD 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. [7]

Arabinose chemical compound

Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde (CHO) functional group.

Cyclic adenosine monophosphate chemical compound

Cyclic adenosine monophosphate is a second messenger important in many biological processes. cAMP is a derivative of adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway. It should not be confused with 5'-AMP-activated protein kinase.

A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes. Bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material. Normally the genes encoding resistance to antibiotics such as ampicillin, chloroamphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli.

Related Research Articles

Lambda phage Bacteriophage

Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate lifecycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase ; mutant strains are unable to lysogenize cells – instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.

Lac repressor DNA-binding protein

The lac repressor is a DNA-binding protein that inhibits the expression of genes coding for proteins involved in the metabolism of lactose in bacteria. These genes are repressed when lactose is not available to the cell, ensuring that the bacterium only invests energy in the production of machinery necessary for uptake and utilization of lactose when lactose is present. When lactose becomes available, it is converted into allolactose, which inhibits the lac repressor's DNA binding ability, thereby increasing gene expression.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

A transcriptional activator is a protein that increases gene transcription of a gene or set of genes. Most activators are DNA-binding proteins that bind to enhancers or promoter-proximal elements.

Basic helix-loop-helix

A basic helix-loop-helix (bHLH) is a protein structural motif that characterizes one of the largest families of dimerizing transcription factors.

Catabolite activator protein

Catabolite activator protein is a trans-acting transcriptional activator that exists as a homodimer in solution. Each subunit of CAP is composed of a ligand-binding domain at the N-terminus and a DNA-binding domain at the C-terminus. Two cAMP molecules bind dimeric CAP with negative cooperativity. Cyclic AMP functions as an allosteric effector by increasing CAP's affinity for DNA. CAP binds a DNA region upstream from the DNA binding site of RNA Polymerase. CAP activates transcription through protein-protein interactions with the α-subunit of RNA Polymerase. This protein-protein interaction is responsible for (i) catalyzing the formation of the RNAP-promoter closed complex; and (ii) isomerization of the RNAP-promoter complex to the open confirmation. CAP's interaction with RNA polymerase causes bending of the DNA near the transcription start site, thus effectively catalyzing the transcription initiation process. CAP's name is derived from its ability to affect transcription of genes involved in many catabolic pathways. For example, when the amount of glucose transported into the cell is low, a cascade of events results in the increase of cytosolic cAMP levels. This increase in cAMP levels is sensed by CAP, which goes on to activate the transcription of many other catabolic genes.

Silencer (genetics) DNA sequence capable of binding repressors

In genetics, a silencer is a DNA sequence capable of binding transcription regulation factors, called repressors. DNA contains genes and provides the template to produce messenger RNA (mRNA). That mRNA is then translated into proteins. When a repressor protein binds to the silencer region of DNA, RNA polymerase is prevented from transcribing the DNA sequence into RNA. With transcription blocked, the translation of RNA into proteins is impossible. Thus, silencers prevent genes from being expressed as proteins.

Regulator gene gene involved in controlling the expression of one or more other genes

A regulator gene, regulator, or regulatory gene is a gene involved in controlling the expression of one or more other genes. Regulatory sequences, which encode regulatory genes, are often at the five prime end (5') to the start site of transcription of the gene they regulate. In addition, these sequences can also be found at the three prime end (3') to the transcription start site. In both cases, whether the regulatory sequence occurs before (5') or after (3') the gene it regulates, the sequence is often many kilobases away from the transcription start site. A regulator gene may encode a protein, or it may work at the level of RNA, as in the case of genes encoding microRNAs. An example of a regulator gene is a gene that codes for a repressor protein that inhibits the activity of an operator.

In molecular biology, an inducer is a molecule that regulates gene expression. An inducer can bind to protein repressors or activators.

Diauxie is a Greek word coined by Jacques Monod to mean two growth phases. The word is used in English in cell biology to describe the growth phases of a microorganism in batch culture as it metabolizes a mixture of two sugars. Rather than metabolizing the two available sugars simultaneously, microbial cells commonly consume them in a sequential pattern, resulting in two separate growth phases.

The galactose permease or GalP found in Escherichia coli is an integral membrane protein involved in the transport of monosaccharides, primarily hexoses, for utilization by E. coli in glycolysis and other metabolic and catabolic pathways (3,4). It is a member of the Major Facilitator Super Family (MFS) and is homologue of the human GLUT1 transporter (4). Below you will find descriptions of the structure, specificity, effects on homeostasis, expression, and regulation of GalP along with examples of several of its homologues.

The L-arabinose operon, also called the ara or araBAD operon, is an operon required for the breakdown of the five-carbon sugar, L-arabinose, in Escherichia coli. The L-arabinose operon contains three structural genes: araB, araA, araD, which encode for three metabolic enzymes that are required for the metabolism of L-arabinose. AraB (ribulokinase), AraA, AraD produced by these genes catalyse conversion of L-arabinose to an intermediate of the pentose phosphate pathway, D-xylulose-5-phosphate.

cAMP receptor protein

cAMP receptor protein is a regulatory protein in bacteria. CRP protein binds cAMP, which causes a conformational change that allows CRP to bind tightly to a specific DNA site in the promoters of the genes it controls. CRP then activates transcription through direct protein–protein interactions with RNA polymerase.

The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. The operon contains two operators, OE and OI. The former is just before the promoter, and the latter is just after the galE gene.

Carbon catabolite repression, or simply catabolite repression, is an important part of global control system of various bacteria and other micro-organisms. Catabolite repression allows micro-organisms to adapt quickly to a preferred carbon and energy source first. This is usually achieved through inhibition of synthesis of enzymes involved in catabolism of carbon sources other than the preferred one. The catabolite repression was first shown to be initiated by glucose and therefore sometimes referred to as the glucose effect. However, the term "glucose effect" is actually a misnomer since other carbon sources are known to induce catabolite repression.

The gua operon is responsible for regulating the synthesis of guanosine mono phosphate(GMP), a purine nucleotide, from inosine monophosphate. It consists of two structural genes guaB(encodes for IMP dehydrogenase or IMPDH) and guaA(encodes for GMP synthetase) apart from the promoter and operator region.

<i>gab</i> operon

The gab operon is responsible for the conversion of γ-aminobutyrate (GABA) to succinate. The gab operon comprises three structural genes – gabD, gabT and gabP – that encode for a succinate semialdehyde dehydrogenase, GABA transaminase and a GABA permease respectively. There is a regulatory gene csiR, downstream of the operon, that codes for a putative transcriptional repressor and is activated when nitrogen is limiting.

References

  1. 1 2 3 4 5 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.
  2. 1 2 3 4 5 6 Schleif R. AraC protein: a love-hate relationship. Bioessays 2003, 25:274-282.
  3. 1 2 3 Reed WL, Schleif RF. Hemiplegic Mutations in AraC Protein. J. Mol. Biol. (1999) 294, 417-425.
  4. 1 2 Lobell RB, Schleif RF. DNA looping and unlooping by AraC protein. Science. 1990 Oct 26;250(4980):528-32.
  5. ↑ 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.
  6. 1 2 Dunn T.M., Schleif R. Deletion Analysis of the Escherichia coli ara PC and PBAD Promoters. J.Mol. Biol. (1984) 180, 201-204.
  7. 1 2 3 4 5 6 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.
  8. ↑ 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).
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.