The gua operon is responsible for regulating the synthesis of guanosine mono phosphate (GMP), a purine nucleotide, from inosine monophosphate (IMP or inosinate). It consists of two structural genes guaB (encodes for IMP dehydrogenase or and guaA (encodes for GMP synthetase) apart from the promoter and operator region.
The first committed step of purine biosynthesis starts from 5-phosphoribosyl 1 pyrophosphate. This undergoes a series of reactions to form IMP, an important branch point in the pathway. The pathway then branches to form adenylosuccinate and then adenylate (AMP) in one branch and xanthylate (XMP) and then guanylate (GMP) in the other branch. IMP dehydrogenase catalyses the conversion of IMP to XMP and GMP synthetase catalyses the conversion of XMP to GMP. [1] [2]
The operon must respond to changes in the metabolic state of the cell. It is subject to growth rate dependent control, stringent control (control during the various stresses the cell is exposed to) and other forms of control. [3] Hence it stops biosynthesis if guanine can be obtained from the external medium, increases its expression if nucleotides are needed (for example during DNA replication) and balances the production of GMP with respect to AMP and the pyrimidine nucleotides. The branch point at IMP mentioned above is tightly controlled rigid node [4] so as to have a balanced production of AMP and GMP. The gua operon is repressed by GMP and is induced by AMP. Similarly AMP synthesis is repressed by AMP itself while it is activated by GMP. This dual control ensures that there is a balance of flux between AMP and GMP and the flux partition remains relatively constant even in the face of perturbations. Some mechanisms by which this control is achieved are discussed below.
Since DNA replication needs a supply of guanine nucleotides, there must be some co-ordination between the DNA replication machinery and the gua operon. One method through which this happens is the DnaA protein. DnaA is a protein which recognises the origin of replication, promotes a local unwinding of an AT rich DNA region and finally guides the helicase DnaB to its entry site. DnaA is the replication initiation factor which causes DNA replication if present in sufficient concentration. [5]
When replication happens, the origin of replication creates a "sink" for DnaA proteins. So genes which are negatively modulated by DnaA, like those of the gua operon are derepressed. Two potential DnaA binding sites, one on the gua promoter and another 200 bp downstream of the IMP dehydrogenase initiation codon on the guaB gene exist. It is thought that both the former and the latter sequences play a part, the latter part being vital and DNA binding at these sequences negatively affect transcription of the gene. [6]
While growing on media in which growth rates are low, cAMP binds to a cAMP receptor protein forming a complex which has regulatory properties. This complex binds to a region 100 bp upstream of the guaB transcription start site which then represses the gua operon. It is a matter of debate of how this complex interacts with RNA polymerase from as long as around 100 bit/s away. One view suggests the involvement of an unknown regulatory factor. In any case, the complex confers growth rate dependent control to the promoter region of the operon. [7]
The repressor purR encoded by purR genes controls the synthesis of enzymes involved in purine biosynthesis. A putative 16 bp pur operator was found in the gua promoter. The purR repressor works with other co-repressors, for example guanine which is a co-repressor in E. coli. [3]
The gua mRNA leader has the potential for forming a stable stem-loop secondary structure incorporating the first 37 nucleotides of the transcript. As the ribosome binding site is sequestered in the stem loop, this structure may be involved in translational regulation. [3]
The gua promoter lies back-to-back with the xseA promoter (encoding the mismatch repair enzyme exonuclease VII). [3] Such close spacing of promoters may have regulatory significance and will lead to steric hindrance as the RNA polymerase molecules try to bind simultaneously. Inactivation of one promoter will naturally lead to greater expression of the other promoter. Other mechanisms include FIS (factor for inversion stimulation) which sterically hinders RNA polymerase binding. But the role of FIS is not yet properly investigated. [8]
Since the metabolic pathway is conserved across species, the genes are similar too. Extensive studies on the regulation in Saccharomyces cerevisiae have been done. The IMD family of genes and gua1 in yeast correspond to guaB and guaA. Here only IMD genes are guanine sensitive and not gua1 unlike in prokaryotes where the entire operon is sensitive. Mycophenolic acid, a drug which is an inhibitor of IMP dehydrogenase, is an inducer of IMD 2 gene (and hence IMD 2 probably has intrinsic drug activity. [9] Another aspect in which the eukaryotic regulation is very different is that eukaryotes have differential regulation of the branches leading to AMP and GMP. For example, in yeasts AMP synthesis genes are poorly repressed by guanine whereas GMP synthesis genes are not affected by adenine and in humans IMP dehydrogenase synthesis is repressed in the presence of guanosine but not adenosine.
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 life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.
Nucleotides are organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules within all life-forms on Earth. Nucleotides are obtained in the diet and are also synthesized from common nutrients by the liver.
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.
In biochemistry, a ribonucleotide is a nucleotide containing ribose as its pentose component. It is considered a molecular precursor of nucleic acids. Nucleotides are the basic building blocks of DNA and RNA. Ribonucleotides themselves are basic monomeric building blocks for RNA. Deoxyribonucleotides, formed by reducing ribonucleotides with the enzyme ribonucleotide reductase (RNR), are essential building blocks for DNA. There are several differences between DNA deoxyribonucleotides and RNA ribonucleotides. Successive nucleotides are linked together via phosphodiester bonds.
The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of β-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 nucleoside triphosphate is a nucleoside containing a nitrogenous base bound to a 5-carbon sugar, with three phosphate groups bound to the sugar. They are the molecular precursors of both DNA and RNA, which are chains of nucleotides made through the processes of DNA replication and transcription. Nucleoside triphosphates also serve as a source of energy for cellular reactions and are involved in signalling pathways.
Biosynthesis, i.e., chemical synthesis occurring in biological contexts, is a term most often referring to multi-step, enzyme-catalyzed processes where chemical substances absorbed as nutrients serve as enzyme substrates, with conversion by the living organism either into simpler or more complex products. Examples of biosynthetic pathways include those for the production of amino acids, lipid membrane components, and nucleotides, but also for the production of all classes of biological macromolecules, and of acetyl-coenzyme A, adenosine triphosphate, nicotinamide adenine dinucleotide and other key intermediate and transactional molecules needed for metabolism. Thus, in biosynthesis, any of an array of compounds, from simple to complex, are converted into other compounds, and so it includes both the catabolism and anabolism of complex molecules. Biosynthetic processes are often represented via charts of metabolic pathways. A particular biosynthetic pathway may be located within a single cellular organelle, while others involve enzymes that are located across an array of cellular organelles and structures.
The trp operon is a group of genes that are transcribed together, encoding the enzymes that produce the amino acid tryptophan in bacteria. The trp operon was first characterized in Escherichia coli, and it has since been discovered in many other bacteria. The operon is regulated so that, when tryptophan is present in the environment, the genes for tryptophan synthesis are repressed.
Nucleic acid metabolism is a collective term that refers to the variety of chemical reactions by which nucleic acids are either synthesized or degraded. Nucleic acids are polymers made up of a variety of monomers called nucleotides. Nucleotide synthesis is an anabolic mechanism generally involving the chemical reaction of phosphate, pentose sugar, and a nitrogenous base. Degradation of nucleic acids is a catabolic reaction and the resulting parts of the nucleotides or nucleobases can be salvaged to recreate new nucleotides. Both synthesis and degradation reactions require multiple enzymes to facilitate the event. Defects or deficiencies in these enzymes can lead to a variety of diseases.
Amino acid biosynthesis is the set of biochemical processes by which the amino acids are produced. The substrates for these processes are various compounds in the organism's diet or growth media. Not all organisms are able to synthesize all amino acids. For example, humans can synthesize 11 of the 20 standard amino acids. These 11 are called the non-essential amino acids.
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, and AraD produced by these genes catalyse conversion of L-arabinose to an intermediate of the pentose phosphate pathway, D-xylulose-5-phosphate.
fis is an E. coli gene encoding the Fis protein. The regulation of this gene is more complex than most other genes in the E. coli genome, as Fis is an important protein which regulates expression of other genes. It is supposed that fis is regulated by H-NS, IHF and CRP. It also regulates its own expression (autoregulation). Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions.
Purine metabolism refers to the metabolic pathways to synthesize and break down purines that are present in many organisms.
Guanosine monophosphate synthetase, also known as GMPS is an enzyme that converts xanthosine monophosphate to guanosine monophosphate.
GMP reductase EC 1.7.1.7 is an enzyme that catalyzes the irreversible and NADPH-dependent reductive deamination of GMP into IMP.
Spot 42 (spf) RNA is a regulatory non-coding bacterial small RNA encoded by the spf gene. Spf is found in gammaproteobacteria and the majority of experimental work on Spot42 has been performed in Escherichia coli and recently in Aliivibrio salmonicida. In the cell Spot42 plays essential roles as a regulator in carbohydrate metabolism and uptake, and its expression is activated by glucose, and inhibited by the cAMP-CRP complex.
The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. Repression of gene expression for this operon works via binding of repressor molecules to two operators. These repressors dimerize, creating a loop in the DNA. The loop as well as hindrance from the external operator prevent RNA polymerase from binding to the promoter, and thus prevent transcription. Additionally, since the metabolism of galactose in the cell is involved in both anabolic and catabolic pathways, a novel regulatory system using two promoters for differential repression has been identified and characterized within the context of the gal operon.
Inosine 5′-monophosphate dehydrogenase (IMPDH) is a purine biosynthetic enzyme that catalyzes the nicotinamide adenine dinucleotide (NAD+)-dependent oxidation of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), the first committed and rate-limiting step towards the de novo biosynthesis of guanine nucleotides from IMP. IMPDH is a regulator of the intracellular guanine nucleotide pool, and is therefore important for DNA and RNA synthesis, signal transduction, energy transfer, glycoprotein synthesis, as well as other process that are involved in cellular proliferation.
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.
PBAD 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. In E. coli, 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.