Regulation of gene expression
Encyclopedia
Gene modulation redirects here. For information on therapeutic regulation of gene expression, see therapeutic gene modulation
.
Regulation of gene expression (or gene regulation) includes the processes that cells
and viruses use to regulate the way that the information in gene
s is turned into gene product
s. Although a functional gene product can be an RNA
, the majority of known mechanisms regulate protein
coding genes. Any step of the gene's expression
may be modulated, from DNA-RNA transcription
to the post-translational modification of a protein.
Gene regulation is essential for viruses, prokaryote
s and eukaryote
s as it increases the versatility and adaptability of an organism
by allowing the cell to express protein when needed. Although in 1951, Barbara McClintock
showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), the first discovery of a gene regulation system is widely considered to be the identification in 1961 of the lac operon
, discovered by Jacques Monod
, in which protein involved in lactose
metabolism are expressed by E. coli
only in the presence of lactose and absence of glucose.
Furthermore, gene regulation drives the processes of cellular differentiation
and morphogenesis
, leading to the creation of different cell types in multicellular organisms where the different types of cells may possess different gene expression profiles though they all possess the same genome
sequence.
step to post-translational modification of a protein. The following is a list of stages where gene expression is regulated, the most extensively utilised point is Transcription Initiation:
is a common method of gene silencing. DNA is typically methylated by methyltransferase enzymes on cytosine nucleotides in a CpG dinucleotide sequence (also called "CpG island
s" when densely clustered). Analysis of the pattern of methylation in a given region of DNA (which can be a promoter) can be achieved through a method called bisulfite mapping. Methylated cytosine residues are unchanged by the treatment, whereas unmethylated ones are changed to uracil. The differences are analyzed by DNA sequencing or by methods developed to quantify SNPs, such as Pyrosequencing
(Biotage) or MassArray (Sequenom
), measuring the relative amounts of C/T at the CG dinucleotide. Abnormal methylation patterns are thought to be involved in oncogenesis.
s are responsible for the amount of supercoiling of DNA, and these complexes can be temporarily modified by processes such as phosphorylation
or more permanently modified by processes such as methylation
. Such modifications are considered to be responsible for more or less permanent changes in gene expression levels.
Histone acetylation is also an important process in transcription. Histone acetyltransferase
enzymes (HATs) such as CREB-binding protein also dissociate the DNA from the histone complex, allowing transcription to proceed. Often, DNA methylation and histone deacetylation work together in gene silencing
. The combination of the two seems to be a signal for DNA to be packed more densely, lowering gene expression.
can be regulated by at least five mechanisms:
s and self-regulate their expression.
), but it is still the most commonly analysed (QPCR and DNA microarray
).
When studying gene expression, there are several methods to look at the various stages. In eukaryotes these include:
Therapeutic gene modulation
Therapeutic gene modulation refers the practice of altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment...
.
- For vocabulary, see Glossary of gene expression termsGlossary of gene expression termsSee also: gene expression, List of Glossaries, List of Natural Sciences Glossaries-A:*Activator - protein that binds to an enhancer and activates transcription from nearby promoter....
Regulation of gene expression (or gene regulation) includes the processes that cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
and viruses use to regulate the way that the information in gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s is turned into gene product
Gene product
A gene product is the biochemical material, either RNA or protein, resulting from expression of a gene. A measurement of the amount of gene product is sometimes used to infer how active a gene is. Abnormal amounts of gene product can be correlated with disease-causing alleles, such as the...
s. Although a functional gene product can be an RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, the majority of known mechanisms regulate protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
coding genes. Any step of the gene's expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
may be modulated, from DNA-RNA transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
to the post-translational modification of a protein.
Gene regulation is essential for viruses, prokaryote
Prokaryote
The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...
s and eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s as it increases the versatility and adaptability of an organism
Organism
In biology, an organism is any contiguous living system . In at least some form, all organisms are capable of response to stimuli, reproduction, growth and development, and maintenance of homoeostasis as a stable whole.An organism may either be unicellular or, as in the case of humans, comprise...
by allowing the cell to express protein when needed. Although in 1951, Barbara McClintock
Barbara McClintock
Barbara McClintock , the 1983 Nobel Laureate in Physiology or Medicine, was an American scientist and one of the world's most distinguished cytogeneticists. McClintock received her PhD in botany from Cornell University in 1927, where she was a leader in the development of maize cytogenetics...
showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), the first discovery of a gene regulation system is widely considered to be the identification in 1961 of the lac operon
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
, discovered by Jacques Monod
Jacques Monod
Jacques Lucien Monod was a French biologist who was awarded a Nobel Prize in Physiology or Medicine in 1965, sharing it with François Jacob and Andre Lwoff "for their discoveries concerning genetic control of enzyme and virus synthesis"...
, in which protein involved in lactose
Lactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...
metabolism are expressed by E. coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
only in the presence of lactose and absence of glucose.
Furthermore, gene regulation drives the processes of cellular differentiation
Cellular differentiation
In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of...
and morphogenesis
Morphogenesis
Morphogenesis , is the biological process that causes an organism to develop its shape...
, leading to the creation of different cell types in multicellular organisms where the different types of cells may possess different gene expression profiles though they all possess the same genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
sequence.
Regulated stages of gene expression
Any step of gene expression may be modulated, from the DNA-RNA transcriptionTranscription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
step to post-translational modification of a protein. The following is a list of stages where gene expression is regulated, the most extensively utilised point is Transcription Initiation:
- Chromatin domainsEpigenomeEpigenome is equivalent to genome in epigenetics. Epigenetics is one of the current topics in cancer research drawing active research. Human tumors undergo a major disruption of DNA methylation and histone modification patterns...
- TranscriptionTranscription (genetics)Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
- Post-transcriptional modificationPost-transcriptional modificationPost-transcriptional modification is a process in cell biology by which, in eukaryotic cells, primary transcript RNA is converted into mature RNA. A notable example is the conversion of precursor messenger RNA into mature messenger RNA , which includes splicing and occurs prior to protein synthesis...
- RNA transport
- TranslationTranslation (genetics)In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
- mRNA degradation
Modification of DNA
In eukaryotes, the accessibility of large regions of DNA can depend on its chromatin structure, which can be altered as a result of histone modifications directed by DNA methylation, ncRNA, or DNA-binding protein. Hence these modifications may up or down regulate the expression of gene. Certain of these modifications that regulate gene expression are inheritable and are referred to as epigenetic regulation.Chemical
Methylation of DNADNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
is a common method of gene silencing. DNA is typically methylated by methyltransferase enzymes on cytosine nucleotides in a CpG dinucleotide sequence (also called "CpG island
CpG island
In genetics, CpG islands or CG islands are genomic regions that contain a high frequency of CpG sites but to date objective definitions for CpG islands are limited. In mammalian genomes, CpG islands are typically 300-3,000 base pairs in length. They are in and near approximately 40% of promoters of...
s" when densely clustered). Analysis of the pattern of methylation in a given region of DNA (which can be a promoter) can be achieved through a method called bisulfite mapping. Methylated cytosine residues are unchanged by the treatment, whereas unmethylated ones are changed to uracil. The differences are analyzed by DNA sequencing or by methods developed to quantify SNPs, such as Pyrosequencing
Pyrosequencing
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides...
(Biotage) or MassArray (Sequenom
Sequenom
Sequenom is a manufacturer of DNA massarrays, based in San Diego, California, United States. The MassARRAY platform is used for SNP genotyping, methylation detection and quantitative gene expression analysis. Sequenom also manufactures clinical tests, such as SEQureDx, a noninvasive prenatal test...
), measuring the relative amounts of C/T at the CG dinucleotide. Abnormal methylation patterns are thought to be involved in oncogenesis.
Structural
Transcription of DNA is dictated by its structure. In general, the density of its packing is indicative of the frequency of transcription. Octameric protein complexes called nucleosomeNucleosome
Nucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool....
s are responsible for the amount of supercoiling of DNA, and these complexes can be temporarily modified by processes such as phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
or more permanently modified by processes such as methylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
. Such modifications are considered to be responsible for more or less permanent changes in gene expression levels.
Histone acetylation is also an important process in transcription. Histone acetyltransferase
Histone acetyltransferase
Histone acetyltransferases are enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl CoA to form ε-N-acetyl lysine....
enzymes (HATs) such as CREB-binding protein also dissociate the DNA from the histone complex, allowing transcription to proceed. Often, DNA methylation and histone deacetylation work together in gene silencing
Gene silencing
Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification...
. The combination of the two seems to be a signal for DNA to be packed more densely, lowering gene expression.
Regulation of transcription
Regulation of transcription controls when transcription occurs and how much RNA is created. Transcription of a gene by RNA polymeraseRNA polymerase
RNA polymerase is an enzyme that produces RNA. In cells, RNAP is needed for constructing RNA chains from DNA genes as templates, a process called transcription. RNA polymerase enzymes are essential to life and are found in all organisms and many viruses...
can be regulated by at least five mechanisms:
- Specificity factorSpecificity factorSpecific Transcription Factors -Eukaryotic transcription begins with the formation of a pre initiation complex formed by the fusion of a group of general transcription factors...
s alter the specificity of RNA polymerase for a given promoter or set of promoters, making it more or less likely to bind to them (i.e., sigma factorSigma factorA sigma factor is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters. Different sigma factors are activated in response to different environmental conditions...
s used in prokaryotic transcriptionProkaryotic transcriptionProkaryotic transcription is the process in which messenger RNA transcripts of genetic material in prokaryotes are produced, to be translated for the production of proteins. Prokaryotic transcription occurs in the cytoplasm alongside translation. Unlike in eukaryotes, prokaryotic transcription and...
). - RepressorRepressorIn molecular genetics, a repressor is a DNA-binding protein that regulates the expression of one or more genes by binding to the operator and blocking the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes. This blocking of expression is called...
s bind to non-coding sequences on the DNA strand that are close to or overlapping the promoter region, impeding RNA polymerase's progress along the strand, thus impeding the expression of the gene. - General transcription factorGeneral transcription factorGeneral transcription factors or basal transcription factors are protein transcription factors that have been shown to be important in the transcription of class II genes to mRNA templates...
s position RNA polymerase at the start of a protein-coding sequence and then release the polymerase to transcribe the mRNA. - ActivatorsActivator (genetics)An activator is a DNA-binding protein that regulates one or more genes by increasing the rate of transcription. The activator may increase transcription by virtue of a connected domain which assists in the formation of the RNA polymerase holoenzyme, or may operate through a coactivator. A...
enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene. Activators do this by increasing the attraction of RNA polymerase for the promoter, through interactions with subunits of the RNA polymerase or indirectly by changing the structure of the DNA. - EnhancersEnhancer (genetics)In genetics, an enhancer is a short region of DNA that can be bound with proteins to enhance transcription levels of genes in a gene cluster...
are sites on the DNA helix that are bound to by activators in order to loop the DNA bringing a specific promoter to the initiation complex. Enhancers are much more common in eukaryote than prokaryotes, where only a few examples exist (to date).
Post-transcriptional regulation
After the DNA is transcribed and mRNA is formed, there must be some sort of regulation on how much the mRNA is translated into proteins. Cells do this by modulating the capping, splicing, addition of a Poly(A) Tail, the sequence-specific nuclear export rates, and, in several contexts, sequestration of the RNA transcript. These processes occur in eukaryotes but not in prokaryotes. This modulation is a result of a protein or transcript that, in turn, is regulated and may have an affinity for certain sequences.Regulation of translation
The translation of mRNA can also be controlled by a number of mechanisms, mostly at the level of initiation. Recruitment of the small ribosomal subunit can indeed be modulated by mRNA secondary structure, antisense RNA binding, or protein binding. In both prokaryotes and eukaryotes, a large number of RNA binding proteins exist, which often are directed to their target sequence by the secondary structure of the transcript, which may change depending on certain conditions, such as temperature or presence of a ligand (aptamer). Some transcripts act as ribozymeRibozyme
A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to catalyze a chemical reaction. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA. Many natural ribozymes catalyze either the hydrolysis of one of their own...
s and self-regulate their expression.
Examples of gene regulation
- Enzyme inductionEnzyme induction and inhibitionEnzyme induction is a process in which a molecule induces the expression of an enzyme.Enzyme inhibition can refer to* the inhibition of the expression of the enzyme by another molecule...
is a process in which a molecule (e.g., a drug) induces (i.e., initiates or enhances) the expression of an enzyme. - The induction of heat shock proteinHeat shock proteinHeat shock proteins are a class of functionally related proteins involved in the folding and unfolding of other proteins. Their expression is increased when cells are exposed to elevated temperatures or other stress. This increase in expression is transcriptionally regulated...
s in the fruit fly Drosophila melanogasterDrosophila melanogasterDrosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
. - The Lac operonLac operonThe lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
is an interesting example of how gene expression can be regulated. - Viruses, despite having only a few genes, possess mechanisms to regulate their gene expression, typically into an early and late phase, using collinear systems regulated by anti-terminators (lambda phageLambda phageEnterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...
) or splicing modulators (HIVHIVHuman immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
).
Developmental biology
A large number of studied regulatory systems come from developmental biology. Examples include:- The colinearity of the Hox gene cluster with their nested antero-posterior patterning
- It has been speculated that pattern generation of the hand (digits - interdigits) The gradient of Sonic hedgehogSonic hedgehogSonic hedgehog homolog is one of three proteins in the mammalian signaling pathway family called hedgehog, the others being desert hedgehog and Indian hedgehog . SHH is the best studied ligand of the hedgehog signaling pathway. It plays a key role in regulating vertebrate organogenesis, such as...
(secreted inducing factor) from the zone of polarizing activityZone of polarizing activityThe Zone of Polarizing Activity is an area of mesenchyme that contains signals which instruct the developing limb bud to form along the anterior/posterior axis. Limb bud is undifferentiated mesenchyme enclosed by an ectoderm covering. Eventually, the limb bud develops into bones, tendons,...
in the limb, which creates a gradient of active Gli3, which activates Gremlin, which inhibits BMPs also secreted in the limb, resulting in the formation of an alternating pattern of activity as a result of this reaction-diffusion system. - Somitogenesis is the creation of segments (somites) from a uniform tissue (Pre-somitic Mesoderm, PSM). They are formed sequentially from anterior to posterior. This is achieved in amniotes possibly by means of two opposing gradients, Retinoic acid in the anterior (wavefront) and Wnt and Fgf in the posterior, coupled to an oscillating pattern (segmentation clock) composed of FGF + Notch and Wnt in antiphase.
- Sex determination in the soma of a Drosophila requires the sensing of the ratio of autosomal genes to sex chromosome-encoded genes, which results in the production of sexless splicing factor in females, resulting in the female isoform of doublesex.
Up-regulation and down-regulation
Up-regulation is a process that occurs within a cell triggered by a signal (originating internal or external to the cell), which results in increased expression of one or more genes and as a result the protein(s) encoded by those genes. On the converse, down-regulation is a process resulting in decreased gene and corresponding protein expression.- Up-regulation occurs, for example, when a cell is deficient in some kind of receptor. In this case, more receptor protein is synthesized and transported to the membrane of the cell and, thus, the sensitivity of the cell is brought back to normal, reestablishing homeostasisHomeostasisHomeostasis is the property of a system that regulates its internal environment and tends to maintain a stable, constant condition of properties like temperature or pH...
.
- Down-regulation occurs, for example, when a cell is overstimulated by a neurotransmitterNeurotransmitterNeurotransmitters are endogenous chemicals that transmit signals from a neuron to a target cell across a synapse. Neurotransmitters are packaged into synaptic vesicles clustered beneath the membrane on the presynaptic side of a synapse, and are released into the synaptic cleft, where they bind to...
, hormoneHormoneA hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
, or drug for a prolonged period of time, and the expression of the receptor protein is decreased in order to protect the cell (see also tachyphylaxisTachyphylaxisTachyphylaxis is a medical term describing a decrease in the response to a drug due to previous exposure to that drug. Increasing the dose of the drug may be able to restore the original response. In this context tachyphylaxis is a synonym for drug tolerance...
).
Inducible vs. repressible systems
Gene Regulation can be summarized by the response of the respective system:- Inducible systems - An inducible system is off unless there is the presence of some molecule (called an inducer) that allows for gene expression. The molecule is said to "induce expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
- Repressible systems - A repressible system is on except in the presence of some molecule (called a corepressor) that suppresses gene expression. The molecule is said to "repress expression". The manner by which this happens is dependent on the control mechanisms as well as differences between prokaryotic and eukaryotic cells.
Theoretical circuits
- Repressor/Inducer: an activation of a sensor results in the change of expression of a gene
- negative feedback: the gene product downregulates its own production directly or indirectly, which can result in
- keeping transcript levels constant/proportional to a factor
- inhibition of run-away reactions when coupled with a positive feedback loop
- creating an oscillator by taking advantage in the time delay of transcription and translation, given that the mRNA and protein half-life is shorter
- positive feedback: the gene product upregulates its own production directly or indirectly, which can result in
- signal amplification
- bistable switches when two genes inhibit each other and both have positive feedback
- pattern generation
Methods
In general, most experiments investigating differential expression used whole cell extracts of RNA, called steady-state levels, to determine which genes changed and by how much they did. These are, however, not informative of where the regulation has occurred and may actually mask conflicting regulatory processess (see post-transcriptional regulationPost-transcriptional regulation
Post-transcriptional regulation is the control of gene expression at the RNA level, therefore between the transcription and the translation of the gene...
), but it is still the most commonly analysed (QPCR and DNA microarray
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
).
When studying gene expression, there are several methods to look at the various stages. In eukaryotes these include:
- The local chromatin environment of the region can be determined by ChIP-chip analysis by pulling down RNA Polymerase IIRNA polymerase IIRNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
, Histone 3 modifications, Trithorax-group protein, Polycomb-group protein, or any other DNA-binding element to which a good antibody is available. - Epistatic interactions can be investigated by synthetic genetic arraySynthetic genetic arraySynthetic Genetic Array analysis is a high-throughput technique for exploring synthetic lethal and synthetic sick genetic interactions . SGA allows for the systematic construction of double mutants using a combination of recombinant genetic techniques, mating and selection steps...
analysis - Due to post-transcriptional regulation, transcription rates and total RNA levels differ significantly. To measure the transcription rates nuclear run-onNuclear run-onA nuclear run-on assay is conducted to identify the genes that are being transcribed at a certain time. Cell nuclei are isolated rapidly, and incubated with labelled nucleotides and the results are hybridized to a slot blot, which is then exposed to film. It was originally developed by Gariglio et...
assays can be done and newer high-throughput methods are being developed, using thiolThiolIn organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl group...
labelling instead of radioactivity. - Only 5% of the RNA polymerised in the nucleus actually exists, and not only introns, abortive products, and non-sense transcripts are degradated. Therefore, the differences in nuclear and cytoplasmic levels can be see by separating the two fractions by gentle lysis.
- Alternative splicing can be analysed with a splicing array or with a tiling array (see DNA microarrayDNA microarrayA DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
). - All in vivoIn vivoIn vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
RNA is complexed as RNPRNP-Medicine:* Ribonucleoprotein, a compound of ribonucleic acid and protein* Ribonucleoprotein Particle, intracellular compartments involved in post-transcriptional fate-Military:* Royal Navy Police, the new name for the British Royal Navy Regulating Branch...
s. The quantity of transcripts bound to specific protein can be also analysed by RIP-ChipRIP-ChipRIP-Chip is immunoprecipitation of an RNA-binding protein coupled to reverse transcription and a microarray. It has been used to find interactions between RNA and protein ....
. For example, DCP2DCP2mRNA-decapping enzyme 2 is a protein that in humans is encoded by the DCP2 gene.-Interactions:DCP2 has been shown to interact with DCP1A and UPF1.-Further reading:...
will give an indication of sequestered protein; ribosomeRibosomeA ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
-bound gives and indication of transcripts active in transcription (although it should be noted that a more dated method, called polysomePolysomePolyribosomes also known as ergosomes are a cluster of ribosomes, bound to a mRNA molecule, first discovered and characterized by Jonathan Warner, Paul Knopf, and Alex Rich in 1963. Many ribosomes read one mRNA simultaneously, progressing along the mRNA to synthesize the same protein...
fractionation, is still popular in some labs) - Protein levels can be analysed by Mass spectrometryMass spectrometryMass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
, which can be compared only to QPCR data, as microarrayDNA microarrayA DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
data is relative and not absolute. - RNA and protein degradation rates are measured by means of transcription inhibitors (actinomycin D or α-amanitin) or translation inhibitors (CycloheximideCycloheximideCycloheximide is an inhibitor of protein biosynthesis in eukaryotic organisms, produced by the bacterium Streptomyces griseus. Cycloheximide exerts its effect by interfering with the translocation step in protein synthesis thus blocking translational elongation...
), respectively.
See also
- Enhancer (genetics)Enhancer (genetics)In genetics, an enhancer is a short region of DNA that can be bound with proteins to enhance transcription levels of genes in a gene cluster...
- Artificial transcription factorArtificial transcription factorAn artificial transcription factor is an example of a chimeral protein, designed to target and modulate gene transcription.They are generally composed of a DNA-binding domain coupled to a modulatory domain in order to alter the expression of a particular gene...
s (small molecules that mimic transcription factor protein) - Cellular modelCellular modelCreating a cellular model has been a particularly challenging task of systems biology and mathematical biology.It involves developing efficient algorithms, data structures, visualization and communication tools to orchestrate the integration of large quantities of biological data with the goal of...
- Conserved non-coding DNA sequenceConserved non-coding sequenceA conserved non-coding sequence is a DNA sequence of noncoding DNA that is evolutionarily conserved. These sequences are of interest for their potential to regulate gene production....
- Spatiotemporal gene expressionSpatiotemporal gene expressionSpatiotemporal gene expression is the activation of genes within specific tissues of an organism at specific times during development. Gene activation patterns vary widely in complexity. Some are straightforward and static, such as the pattern of tubulin, which is expressed in all cells at all...