![Extended Central Dogma with Enzymes](https://upload.wikimedia.org/wikipedia/commons/d/dd/Extended_Central_Dogma_with_Enzymes.jpg)
Gene expression is the process by which information from a
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
is used in the synthesis of a functional
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 ...
that enables it to produce end products, protein or
non-coding RNA
A non-coding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein. The DNA sequence from which a functional non-coding RNA is transcribed is often called an RNA gene. Abundant and functionally important types of non-c ...
, and ultimately affect a
phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
, as the final effect. These products are often
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s, but in non-protein-coding genes such as
transfer RNA (tRNA) and
small nuclear RNA (snRNA), the product is a functional
non-coding RNA
A non-coding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein. The DNA sequence from which a functional non-coding RNA is transcribed is often called an RNA gene. Abundant and functionally important types of non-c ...
. Gene expression is summarized in the
central dogma of molecular biology
The central dogma of molecular biology is an explanation of the flow of genetic information within a biological system. It is often stated as "DNA makes RNA, and RNA makes protein", although this is not its original meaning. It was first stated by ...
first formulated by
Francis Crick
Francis Harry Compton Crick (8 June 1916 – 28 July 2004) was an English molecular biologist, biophysicist, and neuroscientist. He, James Watson, Rosalind Franklin, and Maurice Wilkins played crucial roles in deciphering the helical struc ...
in 1958, further developed in his 1970 article, and expanded by the subsequent discoveries of
reverse transcription
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
and
RNA replication
RNA-dependent RNA polymerase (RdRp) or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template. Specifically, it catalyzes synthesis of the RNA strand complementary to a given RNA template. This is in contrast to t ...
.
The process of gene expression is used by all known life—
eukaryotes
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
(including
multicellular organisms
A multicellular organism is an organism that consists of more than one cell, in contrast to unicellular organism.
All species of animals, land plants and most fungi are multicellular, as are many algae, whereas a few organisms are partially uni- ...
),
prokaryotes
A prokaryote () is a single-celled organism that lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek πρό (, 'before') and κάρυον (, 'nut' or 'kernel').Campbell, N. "Biology:Concepts & Connec ...
(
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
and
archaea
Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
), and utilized by
virus
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
es—to generate the
macromolecular
A macromolecule is a very large molecule important to biophysical processes, such as a protein or nucleic acid. It is composed of thousands of covalently bonded atoms. Many macromolecules are polymers of smaller molecules called monomers. The ...
machinery for life.
In
genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...
, gene expression is the most fundamental level at which the
genotype
The genotype of an organism is its complete set of genetic material. Genotype can also be used to refer to the alleles or variants an individual carries in a particular gene or genetic location. The number of alleles an individual can have in a ...
gives rise to the
phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
, ''i.e.'' observable trait. The genetic information stored in
DNA represents the genotype, whereas the phenotype results from the "interpretation" of that information. Such phenotypes are often displayed by the synthesis of proteins that control the organism's structure and development, or that act as
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
s catalyzing specific metabolic pathways.
All steps in the gene expression process may be modulated (regulated), including the
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
,
RNA splicing
RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns (non-coding regions of RNA) and ''splicing'' b ...
,
translation
Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
, and
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
of a protein.
Regulation of gene expression
Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA). Sophisticated programs of gene expression are wide ...
gives control over the timing, location, and amount of a given gene product (protein or ncRNA) present in a cell and can have a profound effect on the cellular structure and function. Regulation of gene expression is the basis for
cellular differentiation
Cellular differentiation is the process in which a stem cell alters from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular ...
,
development
Development or developing may refer to:
Arts
*Development hell, when a project is stuck in development
*Filmmaking, development phase, including finance and budgeting
*Development (music), the process thematic material is reshaped
*Photographi ...
,
morphogenesis
Morphogenesis (from the Greek ''morphê'' shape and ''genesis'' creation, literally "the generation of form") is the biological process that causes a cell, tissue or organism to develop its shape. It is one of three fundamental aspects of devel ...
and the versatility and
adaptability
Adaptability ( la, adaptō "fit to, adjust") is a feature of a system or of a process. This word has been put to use as a specialised term in different disciplines and in business operations. Word definitions of adaptability as a specialised term d ...
of any
organism
In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and ...
. Gene regulation may therefore serve as a substrate for evolutionary change.
Mechanism
Transcription
![simple transcription elongation1](https://upload.wikimedia.org/wikipedia/commons/2/21/Simple_transcription_elongation1.svg)
The production of a RNA copy from a DNA strand is called
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
, and is performed by
RNA polymerase
In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template.
Using the enzyme helicase, RNAP locally opens the ...
s, which add one ribo
nucleotide
Nucleotides are organic molecules consisting of a nucleoside 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 wi ...
at a time to a growing RNA strand as per the
complementarity law of the nucleotide bases. This RNA is
complementary
A complement is something that completes something else.
Complement may refer specifically to:
The arts
* Complement (music), an interval that, when added to another, spans an octave
** Aggregate complementation, the separation of pitch-class ...
to the template 3′ → 5′ DNA strand,
with the exception that
thymine
Thymine () ( symbol T or Thy) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nu ...
s (T) are replaced with
uracil
Uracil () (symbol U or Ura) is one of the four nucleobases in the nucleic acid RNA. The others are adenine (A), cytosine (C), and guanine (G). In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by ...
s (U) in the RNA.
In prokaryotes, transcription is carried out by a single type of RNA polymerase, which needs to bind a DNA sequence called a
Pribnow box
The Pribnow box (also known as the Pribnow-Schaller box) is a sequence of ''TATAAT'' of six nucleotides (thymine, adenine, thymine, etc.) that is an essential part of a promoter site on DNA for transcription to occur in bacteria.
It is an ideal ...
with the help of the
sigma factor
A sigma factor (σ factor or specificity factor) is a protein needed for initiation of transcription in bacteria. It is a bacterial transcription initiation factor that enables specific binding of RNA polymerase (RNAP) to gene promoters. It is ho ...
protein (σ factor) to start transcription. In eukaryotes, transcription is performed in the nucleus by three types of RNA polymerases, each of which needs a special DNA sequence called the
promoter and a set of DNA-binding proteins—
transcription factors
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The func ...
—to initiate the process (see regulation of transcription below).
RNA polymerase I
RNA polymerase 1 (also known as Pol I) is, in higher eukaryotes, the polymerase that only transcribes ribosomal RNA (but not 5S rRNA, which is synthesized by RNA polymerase III), a type of RNA that accounts for over 50% of the total RNA synthesiz ...
is responsible for transcription of ribosomal RNA (rRNA) genes.
RNA polymerase II
RNA polymerase II (RNAP II and Pol II) is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNAP enzymes found in the nucleus of eukaryoti ...
(Pol II) transcribes all protein-coding genes but also some non-coding RNAs (''e.g.'', snRNAs, snoRNAs or long non-coding RNAs).
RNA polymerase III
In eukaryote cells, RNA polymerase III (also called Pol III) is a protein that transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs.
The genes transcribed by RNA Pol III fall in the category of "housekeeping" genes whose e ...
transcribes 5S rRNA, transfer RNA (tRNA) genes, and some small non-coding RNAs (''e.g.'',
7SK). Transcription ends when the polymerase encounters a sequence called the
terminator
Terminator may refer to:
Science and technology
Genetics
* Terminator (genetics), the end of a gene for transcription
* Terminator technology, proposed methods for restricting the use of genetically modified plants by causing second generation s ...
.
mRNA processing
While transcription of prokaryotic protein-coding genes creates
messenger RNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the p ...
(mRNA) that is ready for translation into protein, transcription of eukaryotic genes leaves a
primary transcript
A primary transcript is the single-stranded ribonucleic acid (RNA) product synthesized by transcription of DNA, and processed to yield various mature RNA products such as mRNAs, tRNAs, and rRNAs. The primary transcripts designated to be mRNAs a ...
of RNA (
pre-RNA), which first has to undergo a series of modifications to become a mature RNA. Types and steps involved in the maturation processes vary between coding and non-coding preRNAs; ''i.e.'' even though preRNA molecules for both mRNA and
tRNA
Transfer RNA (abbreviated tRNA and formerly referred to as sRNA, for soluble RNA) is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes), that serves as the physical link between the mRNA and the amino ac ...
undergo splicing, the steps and machinery involved are different. The processing of non-coding RNA is described below (non-coding RNA maturation).
The processing of premRNA include 5′ ''capping'', which is set of enzymatic reactions that add
7-methylguanosine
7-Methylguanosine (m7G) is a modified purine nucleoside. It is a methylated version of guanosine and when found in human urine, it may be a biomarker of some types of cancer. In the RNAs, 7-methylguanosine have been used to study and examine the ...
(m
7G) to the 5′ end of pre-mRNA and thus protect the RNA from degradation by
exonucleases
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is th ...
. The m
7G cap is then bound by
cap binding complex
The 5' cap of eukaryotic messenger RNA is bound at all times by various cap-binding complexes (CBCs).
Nuclear cap-binding complex
In the nucleus, freshly transcribed mRNA molecules are bound on the 5' cap by the nuclear cap-binding complex of C ...
heterodimer (CBC20/CBC80), which aids in mRNA export to cytoplasm and also protect the RNA from decapping.
Another modification is 3′ ''cleavage and polyadenylation''. They occur if polyadenylation signal sequence (5′- AAUAAA-3′) is present in pre-mRNA, which is usually between protein-coding sequence and terminator. The pre-mRNA is first cleaved and then a series of ~200 adenines (A) are added to form poly(A) tail, which protects the RNA from degradation. The poly(A) tail is bound by multiple
poly(A)-binding proteins (PABPs) necessary for mRNA export and translation re-initiation. In the inverse process of deadenylation, poly(A) tails are shortened by the
CCR4-Not
Carbon Catabolite Repression—Negative On TATA-less, or CCR4-Not, is a multiprotein complex that functions in gene expression. The complex has multiple enzymatic activities as both a poly(A) 3′-5′ exonuclease and a ubiquitin ligase. The co ...
3′-5′ exonuclease, which often leads to full transcript decay.
A very important modification of eukaryotic pre-mRNA is ''
RNA splicing
RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns (non-coding regions of RNA) and ''splicing'' b ...
''. The majority of eukaryotic pre-mRNAs consist of alternating segments called
exons
An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequence ...
and
introns
An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene. ...
. During the process of splicing, an RNA-protein catalytical complex known as
spliceosome
A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to specifi ...
catalyzes two transesterification reactions, which remove an intron and release it in form of lariat structure, and then splice neighbouring exons together. In certain cases, some introns or exons can be either removed or retained in mature mRNA. This so-called
alternative splicing
Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be ...
creates series of different transcripts originating from a single gene. Because these transcripts can be potentially translated into different proteins, splicing extends the complexity of eukaryotic gene expression and the size of a species
proteome
The proteome is the entire set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a certain time. It is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. ...
.
Extensive RNA processing may be an
evolutionary advantage made possible by the nucleus of eukaryotes. In prokaryotes, transcription and translation happen together, whilst in eukaryotes, the
nuclear membrane
The nuclear envelope, also known as the nuclear membrane, is made up of two lipid bilayer membranes that in eukaryotic cells surround the nucleus, which encloses the genetic material.
The nuclear envelope consists of two lipid bilayer membrane ...
separates the two processes, giving time for RNA processing to occur.
Non-coding RNA maturation
In most organisms
non-coding genes (ncRNA) are transcribed as precursors that undergo further processing. In the case of ribosomal RNAs (rRNA), they are often transcribed as a pre-rRNA that contains one or more rRNAs. The pre-rRNA is cleaved and modified (2′-O-methylation and
pseudouridine
Pseudouridine (abbreviated by the Greek letter psi- Ψ) is an isomer of the nucleoside uridine in which the uracil is attached via a carbon-carbon instead of a nitrogen-carbon glycosidic bond. (In this configuration, uracil is sometimes referred ...
formation) at specific sites by approximately 150 different small nucleolus-restricted RNA species, called snoRNAs. SnoRNAs associate with proteins, forming snoRNPs. While snoRNA part basepair with the target RNA and thus position the modification at a precise site, the protein part performs the catalytical reaction. In eukaryotes, in particular a snoRNP called RNase, MRP cleaves the 45S pre-rRNA into the 28S, 5.8S, and 18S rRNAs. The rRNA and RNA processing factors form large aggregates called the
nucleolus
The nucleolus (, plural: nucleoli ) is the largest structure in the nucleus of eukaryotic cells. It is best known as the site of ribosome biogenesis, which is the synthesis of ribosomes. The nucleolus also participates in the formation of sig ...
.
In the case of transfer RNA (tRNA), for example, the 5′ sequence is removed by
RNase P
Ribonuclease P (, ''RNase P'') is a type of ribonuclease which cleaves RNA. RNase P is unique from other RNases in that it is a ribozyme – a ribonucleic acid that acts as a catalyst in the same way that a protein-based enzyme would. Its fu ...
,
whereas the 3′ end is removed by the
tRNase Z enzyme
and the non-templated 3′ CCA tail is added by a
nucleotidyl transferase
Nucleotidyltransferases are transferase enzymes of phosphorus-containing groups, e.g., substituents of nucleotidylic acids or simply nucleoside monophosphates. The general reaction of transferring a nucleoside monophosphate moiety from A to B, can ...
.
In the case of
micro RNA (miRNA), miRNAs are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus by the enzymes
Drosha
Drosha is a Class 2 ribonuclease III enzyme that in humans is encoded by the ''DROSHA'' (formerly ''RNASEN'') gene. It is the primary nuclease that executes the initiation step of miRNA processing in the nucleus. It works closely with DGCR8 and ...
and
Pasha
Pasha, Pacha or Paşa ( ota, پاشا; tr, paşa; sq, Pashë; ar, باشا), in older works sometimes anglicized as bashaw, was a higher rank in the Ottoman Empire, Ottoman political and military system, typically granted to governors, gener ...
. After being exported, it is then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease
Dicer
Dicer, also known as endoribonuclease Dicer or helicase with RNase motif, is an enzyme that in humans is encoded by the gene. Being part of the RNase III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short d ...
, which also initiates the formation of the
RNA-induced silencing complex (RISC), composed of the
Argonaute
The Argonaute protein family, first discovered for its evolutionarily conserved stem cell function, plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). RISC is responsible for the g ...
protein.
Even snRNAs and snoRNAs themselves undergo series of modification before they become part of functional RNP complex. This is done either in the nucleoplasm or in the specialized compartments called
Cajal bodies
Cajal bodies (CBs) also coiled bodies, are spherical nuclear bodies of 0.3–1.0 µm in diameter found in the nucleus of proliferative cells like embryonic cells and tumor cells, or metabolically active cells like neurons. CBs are membrane-le ...
. Their bases are methylated or pseudouridinilated by a group of
small Cajal body-specific RNAs (scaRNAs), which are structurally similar to snoRNAs.
RNA export
In eukaryotes most mature RNA must be exported to the cytoplasm from the
nucleus
Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to:
*Atomic nucleus, the very dense central region of an atom
*Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA
Nucle ...
. While some RNAs function in the nucleus, many RNAs are transported through the
nuclear pore
A nuclear pore is a part of a large complex of proteins, known as a nuclear pore complex that spans the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are approximately 1,000 nuclear pore complexes ...
s and into the
cytosol
The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells (intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...
.
Export of RNAs requires association with specific proteins known as exportins. Specific exportin molecules are responsible for the export of a given RNA type. mRNA transport also requires the correct association with
Exon Junction Complex
An exon junction complex (EJC) is a protein complex which forms on a pre-messenger RNA strand at the junction of two exons which have been joined together during RNA splicing. The EJC has major influences on translation, surveillance and localiza ...
(EJC), which ensures that correct processing of the mRNA is completed before export. In some cases RNAs are additionally transported to a specific part of the cytoplasm, such as a
synapse
In the nervous system, a synapse is a structure that permits a neuron (or nerve cell) to pass an electrical or chemical signal to another neuron or to the target effector cell.
Synapses are essential to the transmission of nervous impulses from ...
; they are then towed by
motor protein
Motor proteins are a class of molecular motors that can move along the cytoplasm of cells. They convert chemical energy into mechanical work by the hydrolysis of ATP. Flagellar rotation, however, is powered by a proton pump.
Cellular functions ...
s that bind through linker proteins to specific sequences (called "zipcodes") on the RNA.
Translation
For some RNA (non-coding RNA) the mature RNA is the final gene product.
In the case of messenger RNA (mRNA) the RNA is an information carrier coding for the synthesis of one or more proteins. mRNA carrying a single protein sequence (common in eukaryotes) is
monocistronic
A cistron is an alternative term for "gene". The word cistron is used to emphasize that genes exhibit a specific behavior in a cis-trans test; distinct positions (or loci) within a genome are cistronic.
History
The words ''cistron'' and ''gene ...
whilst mRNA carrying multiple protein sequences (common in prokaryotes) is known as
polycistronic
A cistron is an alternative term for "gene". The word cistron is used to emphasize that genes exhibit a specific behavior in a cis-trans test; distinct positions (or loci) within a genome are cistronic.
History
The words ''cistron'' and ''gene ...
.
![Ribosome mRNA translation en](https://upload.wikimedia.org/wikipedia/commons/b/b1/Ribosome_mRNA_translation_en.svg)
Every mRNA consists of three parts: a 5′ untranslated region (5′UTR), a protein-coding region or open reading frame (ORF), and a 3′ untranslated region (3′UTR). The coding region carries information for protein synthesis encoded by the
genetic code
The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
to form triplets. Each triplet of nucleotides of the
coding region
The coding region of a gene, also known as the coding sequence (CDS), is the portion of a gene's DNA or RNA that codes for protein. Studying the length, composition, regulation, splicing, structures, and functions of coding regions compared to no ...
is called a
codon
The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
and corresponds to a binding site complementary to an anticodon triplet in transfer RNA. Transfer RNAs with the same anticodon sequence always carry an identical type of
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
. Amino acids are then chained together by the
ribosome
Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to ...
according to the order of triplets in the coding region. The ribosome helps transfer RNA to bind to messenger RNA and takes the amino acid from each transfer RNA and makes a structure-less protein out of it.
Each mRNA molecule is translated into many protein molecules, on average ~2800 in mammals.
In prokaryotes translation generally occurs at the point of transcription (co-transcriptionally), often using a messenger RNA that is still in the process of being created. In eukaryotes translation can occur in a variety of regions of the cell depending on where the protein being written is supposed to be. Major locations are the
cytoplasm
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...
for soluble cytoplasmic proteins and the membrane of the
endoplasmic reticulum
The endoplasmic reticulum (ER) is, in essence, the transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough endoplasmic reticulum ( ...
for proteins that are for export from the cell or insertion into a cell
membrane
A membrane is a selective barrier; it allows some things to pass through but stops others. Such things may be molecules, ions, or other small particles. Membranes can be generally classified into synthetic membranes and biological membranes. B ...
. Proteins that are supposed to be produced at the endoplasmic reticulum are recognised part-way through the translation process. This is governed by the
signal recognition particle
The signal recognition particle (SRP) is an abundant, cytosolic, universally conserved ribonucleoprotein (protein-RNA complex) that recognizes and targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane ...
—a protein that binds to the ribosome and directs it to the endoplasmic reticulum when it finds a
signal peptide
A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16-30 amino acids long) present at the N-ter ...
on the growing (nascent) amino acid chain.
Folding
Each
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
exists as an unfolded
polypeptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
A p ...
or random coil when translated from a sequence of
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
into a linear chain of
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s. This polypeptide lacks any developed three-dimensional structure (the left hand side of the neighboring figure). The polypeptide then folds into its characteristic and functional
three-dimensional structure from a
random coil
In polymer chemistry, a random coil is a conformation of polymers where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the cha ...
.
Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein (the right hand side of the figure) known as the
native state
In biochemistry, the native state of a protein or nucleic acid is its properly folded and/or assembled form, which is operative and functional. The native state of a biomolecule may possess all four levels of biomolecular structure, with the s ...
. The resulting three-dimensional structure is determined by the amino acid sequence (
Anfinsen's dogma
Anfinsen's dogma, also known as the thermodynamic hypothesis, is a postulate in molecular biology. It states that, at least for a small globular protein in its standard physiological environment, the native structure is determined only by the pro ...
).
The correct three-dimensional structure is essential to function, although some parts of functional proteins
may remain unfolded. Failure to fold into the intended shape usually produces inactive proteins with different properties including toxic
prion
Prions are misfolded proteins that have the ability to transmit their misfolded shape onto normal variants of the same protein. They characterize several fatal and transmissible neurodegenerative diseases in humans and many other animals. It ...
s. Several
neurodegenerative
A neurodegenerative disease is caused by the progressive loss of structure or function of neurons, in the process known as neurodegeneration. Such neuronal damage may ultimately involve cell death. Neurodegenerative diseases include amyotrophic ...
and other
disease
A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...
s are believed to result from the accumulation of ''misfolded'' proteins.
Many
allergies
Allergies, also known as allergic diseases, refer a number of conditions caused by the hypersensitivity of the immune system to typically harmless substances in the environment. These diseases include hay fever, food allergies, atopic derma ...
are caused by the folding of the proteins, for the immune system does not produce antibodies for certain protein structures.
Enzymes called
chaperones assist the newly formed protein to attain (
fold into) the 3-dimensional structure it needs to function.
Similarly, RNA chaperones help RNAs attain their functional shapes.
Assisting protein folding is one of the main roles of the endoplasmic reticulum in eukaryotes.
Translocation
Secretory proteins of eukaryotes or prokaryotes must be translocated to enter the secretory pathway. Newly synthesized proteins are directed to the eukaryotic Sec61 or prokaryotic SecYEG translocation channel by
signal peptide
A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16-30 amino acids long) present at the N-ter ...
s. The efficiency of protein secretion in eukaryotes is very dependent on the
signal peptide
A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16-30 amino acids long) present at the N-ter ...
which has been used.
Protein transport
Many proteins are destined for other parts of the cell than the cytosol and a wide range of signalling sequences or
(signal peptides) are used to direct proteins to where they are supposed to be. In prokaryotes this is normally a simple process due to limited compartmentalisation of the cell. However, in eukaryotes there is a great variety of different targeting processes to ensure the protein arrives at the correct organelle.
Not all proteins remain within the cell and many are exported, for example,
digestive enzymes
Digestive enzymes are a group of enzymes that break down polymeric macromolecules into their smaller building blocks, in order to facilitate their absorption into the cells of the body. Digestive enzymes are found in the digestive tracts of anim ...
,
hormone
A hormone (from the Greek participle , "setting in motion") is a class of signaling molecules in multicellular organisms that are sent to distant organs by complex biological processes to regulate physiology and behavior. Hormones are required ...
s and
extracellular matrix
In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide stru ...
proteins. In eukaryotes the export pathway is well developed and the main mechanism for the export of these proteins is translocation to the endoplasmic reticulum, followed by transport via the
Golgi apparatus
The Golgi apparatus (), also known as the Golgi complex, Golgi body, or simply the Golgi, is an organelle found in most eukaryotic cells. Part of the endomembrane system in the cytoplasm, it packages proteins into membrane-bound vesicles ins ...
.
Regulation of gene expression
![Tortie-flame](https://upload.wikimedia.org/wikipedia/commons/3/32/Tortie-flame.jpg)
Regulation of gene expression is the control of the amount and timing of appearance of the functional product of a gene. Control of expression is vital to allow a cell to produce the gene products it needs when it needs them; in turn, this gives cells the flexibility to adapt to a variable environment, external signals, damage to the cell, and other stimuli. More generally, gene regulation gives the cell control over all structure and function, and is the basis for
cellular differentiation
Cellular differentiation is the process in which a stem cell alters from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular ...
,
morphogenesis
Morphogenesis (from the Greek ''morphê'' shape and ''genesis'' creation, literally "the generation of form") is the biological process that causes a cell, tissue or organism to develop its shape. It is one of three fundamental aspects of devel ...
and the versatility and adaptability of any organism.
Numerous terms are used to describe types of genes depending on how they are regulated; these include:
* A constitutive gene is a gene that is transcribed continually as opposed to a facultative gene, which is only transcribed when needed.
* A ''
housekeeping gene
In molecular biology, housekeeping genes are typically constitutive genes that are required for the maintenance of basic cellular function, and are expressed in all cells of an organism under normal and patho-physiological conditions. Although ...
'' is a gene that is required to maintain basic cellular function and so is typically expressed in all cell types of an organism. Examples include
actin
Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of over ...
,
GAPDH
Glyceraldehyde 3-phosphate dehydrogenase (abbreviated GAPDH) () is an enzyme of about 37kDa that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. In addition to this long establishe ...
and
ubiquitin
Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 1980s. Fo ...
. Some housekeeping genes are transcribed at a relatively constant rate and these genes can be used as a reference point in experiments to measure the expression rates of other genes.
* A facultative gene is a gene only transcribed when needed as opposed to a constitutive gene.
* An inducible gene is a gene whose expression is either responsive to environmental change or dependent on the position in the cell cycle.
Any step of gene expression may be modulated, from the DNA-RNA transcription step to
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
of a protein. The stability of the final gene product, whether it is RNA or protein, also contributes to the expression level of the gene—an unstable product results in a low expression level. In general gene expression is regulated through changes
in the number and type of interactions between molecules
that collectively influence transcription of DNA
and translation of RNA.
Some simple examples of where gene expression is important are:
* Control of
insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
expression so it gives a signal for
blood glucose regulation
Blood sugar regulation is the process by which the levels of blood sugar, primarily glucose, are maintained by the body within a narrow range. This tight regulation is referred to as glucose homeostasis. Insulin, which lowers blood sugar, and gl ...
.
*
X chromosome inactivation
X-inactivation (also called Lyonization, after English geneticist Mary Lyon) is a process by which one of the copies of the X chromosome is inactivated in therian female mammals. The inactive X chromosome is silenced by being packaged into a ...
in female
mammals
Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or ...
to prevent an "overdose" of the genes it contains.
*
Cyclin
Cyclin is a family of proteins that controls the progression of a cell through the cell cycle by activating cyclin-dependent kinase (CDK) enzymes or group of enzymes required for synthesis of cell cycle.
Etymology
Cyclins were originally disco ...
expression levels control progression through the eukaryotic
cell cycle
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subs ...
.
Transcriptional regulation
Regulation of transcription can be broken down into three main routes of influence; genetic (direct interaction of a control factor with the gene), modulation interaction of a control factor with the transcription machinery and epigenetic (non-sequence changes in DNA structure that influence transcription).
![Lambda repressor 1LMB](https://upload.wikimedia.org/wikipedia/commons/8/8f/Lambda_repressor_1LMB.png)
Direct interaction with DNA is the simplest and the most direct method by which a protein changes transcription levels. Genes often have several protein binding sites around the coding region with the specific function of regulating transcription. There are many classes of regulatory DNA binding sites known as
enhancers,
insulators and
silencers. The mechanisms for regulating transcription are varied, from blocking key binding sites on the DNA for
RNA polymerase
In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template.
Using the enzyme helicase, RNAP locally opens the ...
to acting as an
activator and promoting transcription by assisting RNA polymerase binding.
The activity of transcription factors is further modulated by intracellular signals causing protein post-translational modification including
phosphorylation
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...
,
acetylation
:
In organic chemistry, acetylation is an organic esterification reaction with acetic acid. It introduces an acetyl group into a chemical compound. Such compounds are termed ''acetate esters'' or simply '' acetates''. Deacetylation is the oppo ...
, or
glycosylation
Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not al ...
. These changes influence a transcription factor's ability to bind, directly or indirectly, to promoter DNA, to recruit RNA polymerase, or to favor elongation of a newly synthesized RNA molecule.
The nuclear membrane in eukaryotes allows further regulation of transcription factors by the duration of their presence in the nucleus, which is regulated by reversible changes in their structure and by binding of other proteins.
Environmental stimuli or endocrine signals
may cause modification of regulatory proteins
eliciting cascades of intracellular signals,
which result in regulation of gene expression.
It has become apparent that there is a significant influence of non-DNA-sequence specific effects on transcription. These effects are referred to as
epigenetic
In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
and involve the higher order structure of DNA, non-sequence specific DNA binding proteins and chemical modification of DNA. In general epigenetic effects alter the accessibility of DNA to proteins and so modulate transcription.
![Nucleosome 1KX5 2](https://upload.wikimedia.org/wikipedia/commons/d/db/Nucleosome_1KX5_2.png)
In eukaryotes the structure of
chromatin
Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
, controlled by the
histone code
The histone code is a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications (known as ''histone marks'') to histone proteins, primarily on their unstructured ends. Together with sim ...
, regulates access to DNA with significant impacts on the expression of genes in
euchromatin
Euchromatin (also called "open chromatin") is a lightly packed form of chromatin ( DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription. Euchromatin stands in contrast to heterochromatin, whic ...
and
heterochromatin
Heterochromatin is a tightly packed form of DNA or '' condensed DNA'', which comes in multiple varieties. These varieties lie on a continue between the two extremes of constitutive heterochromatin and facultative heterochromatin. Both play a role ...
areas.
Enhancers, transcription factors, mediator complex and DNA loops in mammalian transcription
![Regulation of transcription in mammals](https://upload.wikimedia.org/wikipedia/commons/d/d7/Regulation_of_transcription_in_mammals.jpg)
Gene expression in mammals is regulated by many
cis-regulatory element
''Cis''-regulatory elements (CREs) or ''Cis''-regulatory modules (CRMs) are regions of non-coding DNA which regulate the transcription of neighboring genes. CREs are vital components of genetic regulatory networks, which in turn control morphoge ...
s, including
core promoters and promoter-proximal elements that are located near the
transcription start sites of genes,
upstream
Upstream may refer to:
* Upstream (bioprocess)
* ''Upstream'' (film), a 1927 film by John Ford
* Upstream (networking)
* ''Upstream'' (newspaper), a newspaper covering the oil and gas industry
* Upstream (petroleum industry)
* Upstream (software ...
on the DNA (towards the 5' region of the
sense strand
In genetics, a sense strand, or coding strand, is the segment within double-stranded DNA that carries the translatable code in the 5′ to 3′ direction, and which is complementary to the antisense strand of DNA, or template strand, which doe ...
). Other important cis-regulatory modules are localized in DNA regions that are distant from the transcription start sites. These include
enhancers
In genetics, an enhancer is a short (50–1500 bp) region of DNA that can be bound by proteins ( activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcription ...
,
silencers,
insulators
Insulator may refer to:
* Insulator (electricity), a substance that resists electricity
** Pin insulator, a device that isolates a wire from a physical support such as a pin on a utility pole
** Strain insulator, a device that is designed to work ...
and tethering elements.
Enhancers and their associated
transcription factors
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The func ...
have a leading role in the regulation of gene expression.
Enhancers
In genetics, an enhancer is a short (50–1500 bp) region of DNA that can be bound by proteins ( activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcription ...
are genome regions that regulate genes. Enhancers control cell-type-specific gene expression programs, most often by looping through long distances to come in physical proximity with the promoters of their target genes.
Multiple enhancers, each often tens or hundred of thousands of nucleotides distant from their target genes, loop to their target gene promoters and coordinate with each other to control gene expression.
The illustration shows an enhancer looping around to come into proximity with the promoter of a target gene. The loop is stabilized by a dimer of a connector protein (e.g. dimer of
CTCF
Transcriptional repressor CTCF also known as 11-zinc finger protein or CCCTC-binding factor is a transcription factor that in humans is encoded by the ''CTCF'' gene. CTCF is involved in many cellular processes, including transcriptional regulatio ...
or
YY1
YY1 (Yin Yang 1) is a transcriptional repressor protein in humans that is encoded by the YY1 gene.
Function
YY1 is a ubiquitously distributed transcription factor belonging to the GLI-Kruppel class of zinc finger proteins. The protein is invo ...
). One member of the dimer is anchored to its binding motif on the enhancer and the other member is anchored to its binding motif on the promoter (represented by the red zigzags in the illustration).
Several cell function-specific transcription factors (among the about 1,600 transcription factors in a human cell)
generally bind to specific motifs on an enhancer.
A small combination of these enhancer-bound transcription factors, when brought close to a promoter by a DNA loop, govern transcription level of the target gene. Mediator (a complex usually consisting of about 26 proteins in an interacting structure) communicates regulatory signals from enhancer DNA-bound transcription factors directly to the RNA polymerase II (pol II) enzyme bound to the promoter.
Enhancers, when active, are generally transcribed from both strands of DNA with RNA polymerases acting in two different directions, producing two eRNAs as illustrated in the figure.
An inactive enhancer may be bound by an inactive transcription factor. Phosphorylation of the transcription factor may activate it and that activated transcription factor may then activate the enhancer to which it is bound (see small red star representing phosphorylation of transcription factor bound to enhancer in the illustration).
An activated enhancer begins transcription of its RNA before activating transcription of messenger RNA from its target gene.
DNA methylation and demethylation in transcriptional regulation
DNA methylation
DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts t ...
is a widespread mechanism for epigenetic influence on gene expression and is seen in
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
and
eukaryotes
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
and has roles in heritable transcription silencing and transcription regulation. Methylation most often occurs on a cytosine (see Figure). Methylation of cytosine primarily occurs in dinucleotide sequences where a cytosine is followed by a guanine, a
CpG site
The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
. The number of
CpG site
The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
s in the human genome is about 28 million.
Depending on the type of cell, about 70% of the CpG sites have a methylated cytosine.
Methylation of cytosine in DNA has a major role in regulating gene expression. Methylation of CpGs in a promoter region of a gene usually represses gene transcription
while methylation of CpGs in the body of a gene increases expression.
TET enzymes
The TET enzymes are a family of ten-eleven translocation (TET) 5-Methylcytosine, methylcytosine dioxygenases. They are instrumental in DNA demethylation. 5-Methylcytosine (see first Figure) is a methylation, methylated form of the DNA base cytosin ...
play a central role in demethylation of methylated cytosines. Demethylation of CpGs in a gene promoter by
TET enzyme activity increases transcription of the gene.
Transcriptional regulation in learning and memory
![Brain regions involved in memory formation](https://upload.wikimedia.org/wikipedia/commons/0/07/Brain_regions_involved_in_memory_formation.jpg)
In a rat, contextual
fear conditioning
Pavlovian fear conditioning is a behavioral paradigm in which organisms learn to predict aversive events. It is a form of learning in which an aversive stimulus (e.g. an electrical shock) is associated with a particular neutral context (e.g., a r ...
(CFC) is a painful learning experience. Just one episode of CFC can result in a life-long fearful memory.
After an episode of CFC, cytosine methylation is altered in the promoter regions of about 9.17% of all genes in the
hippocampus
The hippocampus (via Latin from Greek , 'seahorse') is a major component of the brain of humans and other vertebrates. Humans and other mammals have two hippocampi, one in each side of the brain. The hippocampus is part of the limbic system, a ...
neuron DNA of a rat.
The
hippocampus
The hippocampus (via Latin from Greek , 'seahorse') is a major component of the brain of humans and other vertebrates. Humans and other mammals have two hippocampi, one in each side of the brain. The hippocampus is part of the limbic system, a ...
is where new memories are initially stored. After CFC about 500 genes have increased transcription (often due to demethylation of CpG sites in a promoter region) and about 1,000 genes have decreased transcription (often due to newly formed 5-methylcytosine at CpG sites in a promoter region). The pattern of induced and repressed genes within neurons appears to provide a molecular basis for forming the first transient memory of this training event in the hippocampus of the rat brain.
Some specific mechanisms guiding new DNA methylations and new
DNA demethylation
For molecular biology in mammals, DNA demethylation causes replacement of 5-methylcytosine (5mC) in a DNA sequence by cytosine (C) (see figure of 5mC and C). DNA demethylation can occur by an active process at the site of a 5mC in a DNA sequenc ...
s in the
hippocampus
The hippocampus (via Latin from Greek , 'seahorse') is a major component of the brain of humans and other vertebrates. Humans and other mammals have two hippocampi, one in each side of the brain. The hippocampus is part of the limbic system, a ...
during memory establishment have been established (see
for summary). One mechanism includes guiding the short isoform of the
TET1 DNA demethylation
For molecular biology in mammals, DNA demethylation causes replacement of 5-methylcytosine (5mC) in a DNA sequence by cytosine (C) (see figure of 5mC and C). DNA demethylation can occur by an active process at the site of a 5mC in a DNA sequenc ...
enzyme, TET1s, to about 600 locations on the genome. The guidance is performed by association of TET1s with
EGR1
EGR-1 (Early growth response protein 1) also known as ZNF268 (zinc finger protein 268) or NGFI-A (nerve growth factor-induced protein A) is a protein that in humans is encoded by the ''EGR1'' gene.
EGR-1 is a mammalian transcription factor. It wa ...
protein, a transcription factor important in memory formation. Bringing TET1s to these locations initiates DNA demethylation at those sites, up-regulating associated genes. A second mechanism involves DNMT3A2, a splice-isoform of
DNA methyltransferase
In biochemistry, the DNA methyltransferase (DNA MTase, DNMT) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl m ...
DNMT3A, which adds methyl groups to cytosines in DNA. This isoform is induced by synaptic activity, and its location of action appears to be determined by
histone
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wr ...
post-translational modifications (a
histone code
The histone code is a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications (known as ''histone marks'') to histone proteins, primarily on their unstructured ends. Together with sim ...
). The resulting new
messenger RNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the p ...
s are then transported by
messenger RNP particles (neuronal granules) to synapses of the neurons, where they can be translated into proteins affecting the activities of synapses.
In particular, the
brain-derived neurotrophic factor
Brain-derived neurotrophic factor (BDNF), or abrineurin, is a protein found in the and the periphery. that, in humans, is encoded by the ''BDNF'' gene. BDNF is a member of the neurotrophin family of growth factors, which are related to the cano ...
gene (''BDNF'') is known as a "learning gene."
After CFC there was upregulation of ''BDNF'' gene expression, related to decreased CpG methylation of certain internal promoters of the gene, and this was correlated with learning.
Transcriptional regulation in cancer
The majority of gene
promoters contain a
CpG island
The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
with numerous
CpG site
The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
s.
When many of a gene's promoter CpG sites are
methylated
In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...
the gene becomes silenced.
Colorectal cancers typically have 3 to 6
driver mutations and 33 to 66
hitchhiker
Hitchhiking (also known as thumbing, autostop or hitching) is a means of transportation that is gained by asking individuals, usually strangers, for a ride in their car or other vehicle. The ride is usually, but not always, free.
Nomads have ...
or passenger mutations.
However, transcriptional silencing may be of more importance than mutation in causing progression to cancer. For example, in colorectal cancers about 600 to 800 genes are transcriptionally silenced by CpG island methylation (see
regulation of transcription in cancer Generally, in progression to cancer, hundreds of genes are silenced or activated. Although silencing of some genes in cancers occurs by mutation, a large proportion of carcinogenic gene silencing is a result of altered DNA methylation (see DNA meth ...
). Transcriptional repression in cancer can also occur by other
epigenetic
In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
mechanisms, such as altered expression of
microRNAs
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miR ...
.
In breast cancer, transcriptional repression of
BRCA1
Breast cancer type 1 susceptibility protein is a protein that in humans is encoded by the ''BRCA1'' () gene. Orthologs are common in other vertebrate species, whereas invertebrate genomes may encode a more distantly related gene. ''BRCA1'' is a h ...
may occur more frequently by over-transcribed microRNA-182 than by hypermethylation of the BRCA1 promoter (see
Low expression of BRCA1 in breast and ovarian cancers).
Post-transcriptional regulation
In eukaryotes, where export of RNA is required before translation is possible, nuclear export is thought to provide additional control over gene expression. All transport in and out of the nucleus is via the
nuclear pore
A nuclear pore is a part of a large complex of proteins, known as a nuclear pore complex that spans the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are approximately 1,000 nuclear pore complexes ...
and transport is controlled by a wide range of
importin
Importin is a type of karyopherin that transports protein molecules from the cell's cytoplasm to the nucleus. It does so by binding to specific recognition sequences, called nuclear localization sequences (NLS).
Importin has two subunits, import ...
and
exportin
Karyopherins are proteins involved in transporting molecules between the cytoplasm and the nucleus of a eukaryotic cell. The inside of the nucleus is called the karyoplasm (or nucleoplasm). Generally, karyopherin-mediated transport occurs through ...
proteins.
Expression of a gene coding for a protein is only possible if the messenger RNA carrying the code survives long enough to be translated. In a typical cell, an RNA molecule is only stable if specifically protected from degradation. RNA degradation has particular importance in regulation of expression in eukaryotic cells where mRNA has to travel significant distances before being translated. In eukaryotes, RNA is stabilised by certain post-transcriptional modifications, particularly the
5′ cap and
poly-adenylated tail.
Intentional degradation of mRNA is used not just as a defence mechanism from foreign RNA (normally from viruses) but also as a route of mRNA ''destabilisation''. If an mRNA molecule has a complementary sequence to a
small interfering RNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating wi ...
then it is targeted for destruction via the RNA interference pathway.
Three prime untranslated regions and microRNAs
Three prime untranslated region
In molecular genetics, the three prime untranslated region (3′-UTR) is the section of messenger RNA (mRNA) that immediately follows the translation termination codon. The 3′-UTR often contains regulatory regions that post-transcriptionally ...
s (3′UTRs) of
messenger RNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the p ...
s (mRNAs) often contain regulatory sequences that post-transcriptionally influence gene expression. Such 3′-UTRs often contain both binding sites for
microRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
s (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3′-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3′-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.
The 3′-UTR often contains
microRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3′-UTRs. Among all regulatory motifs within the 3′-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.
As of 2014, the
miRBase
In bioinformatics, miRBase is a biological database that acts as an archive of microRNA sequences and annotations. As of September 2010 it contained information about 15,172 microRNAs. This number has risen to 38,589 by March 2018. The miRBase re ...
web site, an archive of
miRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
sequences
In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is called t ...
and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target
mRNAs
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the p ...
(affecting expression of several hundred genes).
Friedman et al.
estimate that >45,000 miRNA target sites within human mRNA 3′UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.
Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs.
Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold).
The effects of miRNA dysregulation of gene expression seem to be important in cancer.
For instance, in gastrointestinal cancers, nine miRNAs have been identified as
epigenetically
In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
altered and effective in down regulating DNA repair enzymes.
The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders.
Translational regulation
![Neomycin B C](https://upload.wikimedia.org/wikipedia/commons/4/4f/Neomycin_B_C.svg)
Direct regulation of translation is less prevalent than control of transcription or mRNA stability but is occasionally used. Inhibition of protein translation is a major target for
toxin
A toxin is a naturally occurring organic poison produced by metabolic activities of living cells or organisms. Toxins occur especially as a protein or conjugated protein. The term toxin was first used by organic chemist Ludwig Brieger (1849– ...
s and
antibiotic
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...
s, so they can kill a cell by overriding its normal gene expression control.
Protein synthesis inhibitor
A protein synthesis inhibitor is a compound that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins.
While a broad interpretation of this definition could be used t ...
s include the antibiotic
neomycin
Neomycin is an aminoglycoside antibiotic that displays bactericidal activity against gram-negative aerobic bacilli and some anaerobic bacilli where resistance has not yet arisen. It is generally not effective against gram-positive bacilli and an ...
and the toxin
ricin
Ricin ( ) is a lectin (a carbohydrate-binding protein) and a highly potent toxin produced in the seeds of the castor oil plant, ''Ricinus communis''. The median lethal dose (LD50) of ricin for mice is around 22 micrograms per kilogram of body ...
.
Post-translational modifications
Post-translational modifications (PTMs) are
covalent
A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms ...
modifications to proteins. Like RNA splicing, they help to significantly diversify the proteome. These modifications are usually catalyzed by enzymes. Additionally, processes like covalent additions to amino acid side chain residues can often be reversed by other enzymes. However, some, like the
proteolytic cleavage
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, ...
of the protein backbone, are irreversible.
PTMs play many important roles in the cell.
For example, phosphorylation is primarily involved in activating and deactivating proteins and in signaling pathways.
PTMs are involved in transcriptional regulation: an important function of acetylation and methylation is histone tail modification, which alters how accessible DNA is for transcription.
They can also be seen in the immune system, where glycosylation plays a key role.
One type of PTM can initiate another type of PTM, as can be seen in how
ubiquitination
Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 1980s. Fo ...
tags proteins for degradation through proteolysis.
Proteolysis, other than being involved in breaking down proteins, is also important in activating and deactivating them, and in regulating biological processes such as DNA transcription and cell death.
Measurement
Measuring gene expression is an important part of many
life science
Life is a quality that distinguishes matter that has biological processes, such as signaling and self-sustaining processes, from that which does not, and is defined by the capacity for growth, reaction to stimuli, metabolism, energy t ...
s, as the ability to quantify the level at which a particular gene is expressed within a cell, tissue or organism can provide a lot of valuable information. For example, measuring gene expression can:
* Identify viral infection of a cell (
viral protein
A viral protein is both a component and a product of a virus. Viral proteins are grouped according to their functions, and groups of viral proteins include structural proteins, nonstructural proteins, regulatory proteins, and accessory proteins. Vi ...
expression).
* Determine an individual's susceptibility to
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
(
oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels. expression).
* Find if a bacterium is resistant to
penicillin
Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...
(
beta-lactamase
Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem), although carbapenems ...
expression).
Similarly, the analysis of the location of protein expression is a powerful tool, and this can be done on an organismal or cellular scale. Investigation of localization is particularly important for the study of
development
Development or developing may refer to:
Arts
*Development hell, when a project is stuck in development
*Filmmaking, development phase, including finance and budgeting
*Development (music), the process thematic material is reshaped
*Photographi ...
in multicellular organisms and as an indicator of protein function in single cells. Ideally, measurement of expression is done by detecting the final gene product (for many genes, this is the protein); however, it is often easier to detect one of the precursors, typically
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
and to infer gene-expression levels from these measurements.
mRNA quantification
Levels of mRNA can be quantitatively measured by
northern blotting
The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.K ...
, which provides size and sequence information about the mRNA molecules. A sample of RNA is separated on an
agarose gel
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the t ...
and hybridized to a radioactively labeled RNA probe that is complementary to the target sequence. The radiolabeled RNA is then detected by an
autoradiograph
An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance. Alternatively, the autoradiograph is also available ...
. Because the use of radioactive reagents makes the procedure time-consuming and potentially dangerous, alternative labeling and detection methods, such as digoxigenin and biotin chemistries, have been developed. Perceived disadvantages of Northern blotting are that large quantities of RNA are required and that quantification may not be completely accurate, as it involves measuring band strength in an image of a gel. On the other hand, the additional mRNA size information from the Northern blot allows the discrimination of alternately spliced transcripts.
Another approach for measuring mRNA abundance is RT-qPCR. In this technique,
reverse transcription
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
is followed by
quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
. Reverse transcription first generates a DNA template from the mRNA; this single-stranded template is called
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
. The cDNA template is then amplified in the quantitative step, during which the
fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
emitted by labeled
hybridization probe
In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RN ...
s or
intercalating dyes changes as the
DNA amplification Gene amplification refers to a number of natural and artificial processes by which the number of copies of a gene is increased "without a proportional increase in other genes".
Artificial DNA amplification
In research or diagnosis DNA amplificati ...
process progresses. With a carefully constructed standard curve, qPCR can produce an absolute measurement of the number of copies of original mRNA, typically in units of copies per nanolitre of homogenized tissue or copies per cell. qPCR is very sensitive (detection of a single mRNA molecule is theoretically possible), but can be expensive depending on the type of reporter used; fluorescently labeled oligonucleotide probes are more expensive than non-specific intercalating fluorescent dyes.
For
expression profiling
In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
, or high-throughput analysis of many genes within a sample,
quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
may be performed for hundreds of genes simultaneously in the case of low-density arrays. A second approach is the
hybridization microarray. A single array or "chip" may contain probes to determine transcript levels for every known gene in the genome of one or more organisms. Alternatively, "tag based" technologies like
Serial analysis of gene expression
Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those tra ...
(SAGE) and
RNA-Seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
, which can provide a relative measure of the cellular
concentration
In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', ''molar concentration'', ''number concentration'', an ...
of different mRNAs, can be used. An advantage of tag-based methods is the "open architecture", allowing for the exact measurement of any transcript, with a known or unknown sequence. Next-generation sequencing (NGS) such as
RNA-Seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
is another approach, producing vast quantities of sequence data that can be matched to a reference genome. Although NGS is comparatively time-consuming, expensive, and resource-intensive, it can identify
single-nucleotide polymorphisms
In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
, splice-variants, and novel genes, and can also be used to profile expression in organisms for which little or no sequence information is available.
RNA profiles in Wikipedia
![PBB GE SLC2A4 206603 at fs](https://upload.wikimedia.org/wikipedia/commons/7/7f/PBB_GE_SLC2A4_206603_at_fs.png)
Profiles like these are found for almost all proteins listed in Wikipedia. They are generated by organizations such as the
Genomics Institute of the Novartis Research Foundation
Novartis AG is a Swiss-American multinational pharmaceutical corporation based in Basel, Switzerland and
Cambridge, Massachusetts, United States (global research).name="novartis.com">https://www.novartis.com/research-development/research-loc ...
and the
European Bioinformatics Institute
The European Bioinformatics Institute (EMBL-EBI) is an Intergovernmental Organization (IGO) which, as part of the European Molecular Biology Laboratory (EMBL) family, focuses on research and services in bioinformatics. It is located on the Well ...
. Additional information can be found by searching their databases (for an example of the GLUT4 transporter pictured here, see citation). These profiles indicate the level of DNA expression (and hence RNA produced) of a certain protein in a certain tissue, and are color-coded accordingly in the images located in the Protein Box on the right side of each Wikipedia page.
Protein quantification
For genes encoding proteins, the expression level can be directly assessed by a number of methods with some clear analogies to the techniques for mRNA quantification.
One of the most commonly used methods is to perform a
Western blot
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...
against the protein of interest. This gives information on the size of the protein in addition to its identity. A sample (often cellular
lysate
Lysis ( ) is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" ) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a ''lysate''. In molecular bio ...
) is separated on a
polyacrylamide gel
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their ...
, transferred to a membrane and then probed with an
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
to the protein of interest. The antibody can either be conjugated to a
fluorophore
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
or to
horseradish peroxidase
The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various or ...
for imaging and/or quantification. The gel-based nature of this assay makes quantification less accurate, but it has the advantage of being able to identify later modifications to the protein, for example proteolysis or ubiquitination, from changes in size.
mRNA-protein correlation
While transcription directly reflects gene expression, the copy number of mRNA molecules does not directly correlate with the number of protein molecules translated from mRNA. Quantification of both protein and mRNA permits a correlation of the two levels. Regulation on each step of gene expression can impact the correlation, as shown for regulation of translation
or protein stability.
Post-translational factors, such as protein transport in highly polar cells,
can influence the measured mRNA-protein correlation as well.
Localisation
![Hunchback in situ](https://upload.wikimedia.org/wikipedia/commons/f/f7/Hunchback_in_situ.jpg)
Analysis of expression is not limited to quantification; localisation can also be determined. mRNA can be detected with a suitably labelled complementary mRNA strand and protein can be detected via labelled antibodies. The probed sample is then observed by microscopy to identify where the mRNA or protein is.
![GFP structure](https://upload.wikimedia.org/wikipedia/commons/e/e4/GFP_structure.png)
By replacing the gene with a new version fused to a
green fluorescent protein
The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...
(or similar) marker, expression may be directly quantified in live cells. This is done by imaging using a
fluorescence microscope
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microsc ...
. It is very difficult to clone a GFP-fused protein into its native location in the genome without affecting expression levels so this method often cannot be used to measure endogenous gene expression. It is, however, widely used to measure the expression of a gene artificially introduced into the cell, for example via an
expression vector
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
. It is important to note that by fusing a target protein to a fluorescent reporter the protein's behavior, including its cellular localization and expression level, can be significantly changed.
The
enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
works by using antibodies immobilised on a
microtiter plate
A microplate, also known as a microtiter plate (''Microtiter'' is a registered trademark in the United States, therefore it should not be used generically without attribution), microwell plate or multiwell, is a flat plate with multiple "wells" ...
to capture proteins of interest from samples added to the well. Using a detection antibody conjugated to an enzyme or fluorophore the quantity of bound protein can be accurately measured by
fluorometric
Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electron ...
or
colourimetric detection. The detection process is very similar to that of a Western blot, but by avoiding the gel steps more accurate quantification can be achieved.
Expression system
An expression system is a system specifically designed for the production of a gene product of choice. This is normally a protein although may also be RNA, such as
tRNA
Transfer RNA (abbreviated tRNA and formerly referred to as sRNA, for soluble RNA) is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes), that serves as the physical link between the mRNA and the amino ac ...
or a
ribozyme
Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonst ...
. An expression system consists of a gene, normally encoded by
DNA, and the
molecular machinery
A molecular machine, nanite, or nanomachine is a molecular component that produces quasi-mechanical movements (output) in response to specific stimuli (input). In cellular biology, macromolecular machines frequently perform tasks essential for l ...
required to
transcribe
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
the DNA into
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
and
translate
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transl ...
the mRNA into
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
using the reagents provided. In the broadest sense this includes every living cell but the term is more normally used to refer to expression as a laboratory tool. An expression system is therefore often artificial in some manner. Expression systems are, however, a fundamentally natural process. Viruses are an excellent example where they replicate by using the host cell as an expression system for the viral proteins and genome.
Inducible expression
Doxycycline
Doxycycline is a broad-spectrum tetracycline class antibiotic used in the treatment of infections caused by bacteria and certain parasites. It is used to treat bacterial pneumonia, acne, chlamydia infections, Lyme disease, cholera, typhus, an ...
is also used in "Tet-on" and "Tet-off"
tetracycline controlled transcriptional activation
Tetracycline-controlled transcriptional activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline).
Tetracycl ...
to regulate
transgene
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
expression in organisms and
cell culture
Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...
s.
In nature
In addition to these biological tools, certain naturally observed configurations of DNA (genes, promoters, enhancers, repressors) and the associated machinery itself are referred to as an expression system. This term is normally used in the case where a gene or set of genes is switched on under well defined conditions, for example, the simple repressor switch expression system in
Lambda phage
''Enterobacteria phage λ'' (lambda phage, coliphage λ, officially ''Escherichia virus Lambda'') is a bacterial virus, or bacteriophage, that infects the bacterial species ''Escherichia coli'' (''E. coli''). It was discovered by Esther Lederb ...
and the
lac operator system in bacteria. Several natural expression systems are directly used or modified and used for artificial expression systems such as the
Tet-on and Tet-off expression system.
Gene networks
Genes have sometimes been regarded as nodes in a network, with inputs being proteins such as
transcription factor
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
s, and outputs being the level of gene expression. The node itself performs a function, and the operation of these functions have been interpreted as performing a kind of
information processing
Information processing is the change (processing) of information in any manner detectable by an observer. As such, it is a process that ''describes'' everything that happens (changes) in the universe, from the falling of a rock (a change in posit ...
within cells and determines cellular behavior.
Gene networks can also be constructed without formulating an explicit causal model. This is often the case when assembling networks from large expression data sets. Covariation and correlation of expression is computed across a large sample of cases and measurements (often
transcriptome
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The t ...
or
proteome
The proteome is the entire set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a certain time. It is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. ...
data). The source of variation can be either experimental or natural (observational). There are several ways to construct gene expression networks, but one common approach is to compute a matrix of all pair-wise correlations of expression across conditions, time points, or individuals and convert the matrix (after thresholding at some cut-off value) into a graphical representation in which nodes represent genes, transcripts, or proteins and edges connecting these nodes represent the strength of association (se
GeneNetwork GeneNetwork 2.
Techniques and tools
The following experimental techniques are used to measure gene expression and are listed in roughly chronological order, starting with the older, more established technologies. They are divided into two groups based on their degree of
multiplexity.
* Low-to-mid-plex techniques:
**
Reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...
**
Northern blot
The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.K ...
**
Western blot
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...
**
Fluorescent in situ hybridization
Fluorescence ''in situ'' hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed ...
**
Reverse transcription PCR
* Higher-plex techniques:
**
SAGE
Sage or SAGE may refer to:
Plants
* ''Salvia officinalis'', common sage, a small evergreen subshrub used as a culinary herb
** Lamiaceae, a family of flowering plants commonly known as the mint or deadnettle or sage family
** ''Salvia'', a large ...
**
DNA microarray
A DNA microarray (also commonly known as DNA chip or biochip) 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 ...
**
Tiling array
Tiling arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface.
Tiling arrays differ from traditional microarrays in the nature ...
**
RNA-Seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
Gene expression databases
Gene expression omnibus(GEO) at
NCBI
The National Center for Biotechnology Information (NCBI) is part of the United States National Library of Medicine (NLM), a branch of the National Institutes of Health (NIH). It is approved and funded by the government of the United States. The ...
Expression Atlasat the
EBI
Ebrahim Hamedi ( fa, اِبراهیم حامدی, also Romanized as "Ebrāhim Hāmedi"; born 1949), better known by his stage name Ebi (Persian: ), is an Iranian pop singer who first started his career in Tehran, gaining fame as part of a ban ...
* Mous
Gene Expression Databaseat the
Jackson Laboratory
The Jackson Laboratory (often abbreviated as JAX) is an independent, non-profit biomedical research institution which was founded by a eugenicist. It employs more than 3,000 employees in Bar Harbor, Maine; Sacramento, California; Farmington, Con ...
*
CollecTF
CollecTF is a database of transcription factor binding sites in the Bacteria domain.
CollecTF compiles ''only'' experimentally validated TF-binding sites. This is accomplished through the manual curation of peer-reviewed literature with a specia ...
: a database of experimentally validated transcription factor-binding sites in Bacteria.
* COLOMBOS: collection of bacterial expression compendia.
Many Microbe Microarrays Database microbial Affymetrix data
See also
References
External links
Plant Transcription Factor Database and Plant Transcriptional Regulation Data and Analysis Platform
{{DEFAULTSORT:Gene Expression
Molecular biology