![MRNA](https://upload.wikimedia.org/wikipedia/commons/9/9b/MRNA.svg)
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode
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, res ...
s are said to produce
messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called
non-coding RNA
A non-coding RNA (ncRNA) is a functional RNA molecule that is not Translation (genetics), 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 im ...
s (ncRNAs). mRNA comprises only 1–3% of total RNA samples. Less than 2% of the human genome can be transcribed into mRNA (
Human genome#Coding vs. noncoding DNA), while at least 80% of mammalian genomic DNA can be actively transcribed (in one or more types of cells), with the majority of this 80% considered to be ncRNA.
Both DNA and RNA are
nucleic acids, which use
base pairs of
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 biomolecule ...
s as a
complementary language. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary,
antiparallel RNA strand called 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 ...
.
Transcription proceeds in the following general steps:
# RNA polymerase, together with one or more
general transcription factors, binds to
promoter DNA.
# RNA polymerase generates a
transcription bubble
A transcription bubble is a molecular structure formed during DNA transcription when a limited portion of the DNA double helix is unwound. The size of a transcription bubble ranges from 12-14 base pairs. A transcription bubble is formed when the ...
, which separates the two strands of the DNA helix. This is done by breaking the
hydrogen bonds between complementary DNA nucleotides.
# RNA polymerase adds RNA
nucleotides
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 with ...
(which are complementary to the nucleotides of one DNA strand).
# RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.
# Hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand.
# If the cell has a
nucleus, the RNA may be further processed. This may include
polyadenylation,
capping, and
splicing.
# The RNA may remain in the nucleus or exit to 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. ...
through the
nuclear pore complex.
If the stretch of DNA is transcribed into an RNA molecule that encodes a
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, res ...
, the RNA is termed
messenger RNA (mRNA); the mRNA, in turn, serves as a template for the protein's synthesis through
translation. Other stretches of DNA may be transcribed into small
non-coding RNA
A non-coding RNA (ncRNA) is a functional RNA molecule that is not Translation (genetics), 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 im ...
s such as
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. mi ...
,
transfer RNA (tRNA),
small nucleolar RNA (snoRNA),
small nuclear RNA
Small nuclear RNA (snRNA) is a class of small RNA molecules that are found within the splicing speckles and Cajal bodies of the cell nucleus in eukaryotic cells. The length of an average snRNA is approximately 150 nucleotides. They are transcribe ...
(snRNA), or enzymatic RNA molecules called
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 demons ...
s
[Eldra P. Solomon, Linda R. Berg, Diana W. Martin. ''Biology, 8th Edition, International Student Edition''. Thomson Brooks/Cole. ] as well as larger non-coding RNAs such as
ribosomal RNA (rRNA), and
long non-coding RNA (lncRNA). Overall, RNA helps synthesize, regulate, and process proteins; it therefore plays a fundamental role in performing functions within a cell.
In
virology
Virology is the scientific study of biological viruses. It is a subfield of microbiology that focuses on their detection, structure, classification and evolution, their methods of infection and exploitation of host cells for reproduction, th ...
, the term transcription may also be used when referring to mRNA synthesis from an RNA molecule (i.e., equivalent to RNA replication). For instance, the
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding g ...
of a negative-
sense single-stranded RNA (ssRNA -) virus may be a template for a positive-sense single-stranded RNA (ssRNA +). This is because the positive-sense strand contains the sequence information needed to translate the viral proteins needed for
viral replication. This process is catalyzed by a viral
RNA replicase
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 ...
.
Background
A DNA transcription unit encoding for a protein may contain both a ''coding sequence'', which will be translated into the protein, and ''regulatory sequences'', which direct and regulate the synthesis of that protein. The regulatory sequence before ("
upstream" from) the coding sequence is called the
five prime untranslated region
The 5′ untranslated region (also known as 5′ UTR, leader sequence, transcript leader, or leader RNA) is the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon. This region is important for the regulation of t ...
(5'UTR); the sequence after ("
downstream
Downstream may refer to:
* Downstream (bioprocess)
* Downstream (manufacturing)
* Downstream (networking)
* Downstream (software development)
* Downstream (petroleum industry)
* Upstream and downstream (DNA), determining relative positions on DNA ...
" from) the coding sequence is called the
three prime untranslated region (3'UTR).
As opposed to
DNA replication, transcription results in an RNA complement that includes the nucleotide
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 b ...
(U) in all instances where
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 pyrimidi ...
(T) would have occurred in a DNA complement.
Only one of the two DNA strands serve as a template for transcription. The
antisense
In molecular biology and genetics, the sense of a nucleic acid molecule, particularly of a strand of DNA or RNA, refers to the nature of the roles of the strand and its complement in specifying a sequence of amino acids. Depending on the context ...
strand of DNA is read by RNA polymerase from the 3' end to the 5' end during transcription (3' → 5'). The complementary RNA is created in the opposite direction, in the 5' → 3' direction, matching the sequence of the sense strand with the exception of switching uracil for thymine. This directionality is because RNA polymerase can only add nucleotides to the 3' end of the growing mRNA chain. This use of only the 3' → 5' DNA strand eliminates the need for the
Okazaki fragment
Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA ...
s that are seen in DNA replication.
This also removes the need for an
RNA primer to initiate RNA synthesis, as is the case in DNA replication.
The ''non''-template (sense) strand of DNA is called the
coding strand, because its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine). This is the strand that is used by convention when presenting a DNA sequence.
Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA. As a result, transcription has a lower copying fidelity than DNA replication.
Major steps
Transcription is divided into ''initiation'', ''promoter escape'', ''elongation,'' and ''termination''.
Setting up for transcription
Enhancers, transcription factors, Mediator complex and DNA loops in mammalian transcription
Setting up for transcription 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 promoter and promoter-proximal elements that are located near the
transcription start sites of genes. Core promoters combined with
general transcription factor
General transcription factors (GTFs), also known as basal transcriptional factors, are a class of protein transcription factors that bind to specific sites ( promoter) on DNA to activate transcription of genetic information from DNA to messenger ...
s are sufficient to direct transcription initiation, but generally have low basal activity.
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 transcriptio ...
,
silencers,
insulators and tethering elements.
Among this constellation of 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 fun ...
have a leading role in the initiation of gene transcription.
An enhancer localized in a DNA region distant from the promoter of a gene can have a very large effect on gene transcription, with some genes undergoing up to 100-fold increased transcription due to an activated enhancer.
Enhancers are regions of the genome that are major gene-regulatory elements. Enhancers control cell-type-specific gene transcription programs, most often by looping through long distances to come in physical proximity with the promoters of their target genes.
While there are hundreds of thousands of enhancer DNA regions,
for a particular type of tissue only specific enhancers are brought into proximity with the promoters that they regulate. In a study of brain cortical neurons, 24,937 loops were found, bringing enhancers to their target promoters.
Multiple enhancers, each often at tens or hundred of thousands of nucleotides distant from their target genes, loop to their target gene promoters and can coordinate with each other to control transcription of their common target gene.
The schematic illustration in this section shows an enhancer looping around to come into close physical 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), with one member of the dimer anchored to its binding motif on the enhancer and the other member anchored to its binding motif on the promoter (represented by the red zigzags in the illustration).
Several cell function specific transcription factors (there are about 1,600 transcription factors in a human cell
) generally bind to specific motifs on an enhancer
and a small combination of these enhancer-bound transcription factors, when brought close to a promoter by a DNA loop, govern level of transcription 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
enhancer RNAs (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.
CpG island methylation and demethylation
![Cytosine and 5-methylcytosine](https://upload.wikimedia.org/wikipedia/commons/b/b9/Cytosine_and_5-methylcytosine.svg)
Transcription regulation at about 60% of promoters is also controlled by methylation of cytosines within CpG dinucleotides (where 5’ cytosine is followed by 3’ guanine or
CpG sites
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 ...
).
5-methylcytosine (5-mC) is a
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 ...
form of the
DNA base
cytosine (see Figure). 5-mC is an
epigenetic marker found predominantly within CpG sites. About 28 million CpG dinucleotides occur in the human genome.
In most tissues of mammals, on average, 70% to 80% of CpG cytosines are methylated (forming 5-methylCpG or 5-mCpG).
Methylated cytosines within 5’cytosine-guanine 3’ sequences often occur in groups, called
CpG islands. About 60% of promoter sequences have a CpG island while only about 6% of enhancer sequences have a CpG island.
CpG islands constitute regulatory sequences, since if CpG islands are methylated in the promoter of a gene this can reduce or silence gene transcription.
DNA methylation regulates gene transcription through interaction with methyl binding domain (MBD) proteins, such as MeCP2, MBD1 and MBD2. These
MBD proteins bind most strongly to highly methylated
CpG islands.
These MBD proteins have both a methyl-CpG-binding domain as well as a transcription repression domain.
They bind to methylated DNA and guide or direct protein complexes with chromatin remodeling and/or histone modifying activity to methylated CpG islands. MBD proteins generally repress local chromatin such as by catalyzing the introduction of repressive histone marks, or creating an overall repressive chromatin environment through nucleosome remodeling and chromatin reorganization.
As noted in the previous section,
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 fun ...
are proteins that bind to specific DNA sequences in order to regulate the expression of a gene. The binding sequence for a transcription factor in DNA is usually about 10 or 11 nucleotides long. As summarized in 2009, Vaquerizas et al. indicated there are approximately 1,400 different transcription factors encoded in the human genome by genes that constitute about 6% of all human protein encoding genes.
About 94% of transcription factor binding sites (TFBSs) that are associated with signal-responsive genes occur in enhancers while only about 6% of such TFBSs occur in promoters.
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 is a particular transcription factor that is important for regulation of methylation of CpG islands. An
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 ...
transcription factor binding site is frequently located in enhancer or promoter sequences.
There are about 12,000 binding sites for EGR1 in the mammalian genome and about half of EGR1 binding sites are located in promoters and half in enhancers.
The binding of EGR1 to its target DNA binding site is insensitive to cytosine methylation in the DNA.
While only small amounts of EGR1 transcription factor protein are detectable in cells that are un-stimulated, translation of the ''EGR1'' gene into protein at one hour after stimulation is drastically elevated.
Production of EGR1 transcription factor proteins, in various types of cells, can be stimulated by growth factors, neurotransmitters, hormones, stress and injury.
In the brain, when neurons are activated, EGR1 proteins are up-regulated and they bind to (recruit) the pre-existing
TET1 enzymes that are produced in high amounts in neurons.
TET enzymes
The TET enzymes are a family of ten-eleven translocation (TET) methylcytosine dioxygenases. They are instrumental in DNA demethylation. 5-Methylcytosine (see first Figure) is a methylated form of the DNA base cytosine (C) that often regulates ge ...
can catalyse demethylation of 5-methylcytosine. When EGR1 transcription factors bring TET1 enzymes to EGR1 binding sites in promoters, the TET enzymes can
demethylate Demethylating agents are chemical substances that can inhibit methylation, resulting in the expression of the previously hypermethylated silenced genes (see Methylation#Cancer for more detail). Cytidine analogs such as 5-azacytidine (azacitidine) ...
the methylated CpG islands at those promoters. Upon demethylation, these promoters can then initiate transcription of their target genes. Hundreds of genes in neurons are differentially expressed after neuron activation through EGR1 recruitment of TET1 to methylated regulatory sequences in their promoters.
The methylation of promoters is also altered in response to signals. The three mammalian
DNA methyltransferasess (DNMT1, DNMT3A, and DNMT3B) catalyze the addition of methyl groups to cytosines in DNA. While DNMT1 is a “maintenance” methyltransferase, DNMT3A and DNMT3B can carry out new methylations. There are also two
splice protein isoform
A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isof ...
s produced from the ''DNMT3A'' gene: DNA methyltransferase proteins DNMT3A1 and DNMT3A2.
The splice isoform DNMT3A2 behaves like the product of a classical immediate-early gene and, for instance, it is robustly and transiently produced after neuronal activation.
Where the DNA methyltransferase isoform DNMT3A2 binds and adds methyl groups to cytosines appears to be determined by histone post translational modifications.
On the other hand, neural activation causes degradation of DNMT3A1 accompanied by reduced methylation of at least one evaluated targeted promoter.
Initiation
Transcription begins with the binding of RNA polymerase, together with one or more
general transcription factors, to a specific DNA sequence referred to as a "
promoter" to form an RNA polymerase-promoter "closed complex". In the "closed complex" the promoter DNA is still fully double-stranded.
RNA polymerase, assisted by one or more general transcription factors, then unwinds approximately 14 base pairs of DNA to form an RNA polymerase-promoter "open complex". In the "open complex" the promoter DNA is partly unwound and single-stranded. The exposed, single-stranded DNA is referred to as the "transcription bubble."
RNA polymerase, assisted by one or more general transcription factors, then selects a transcription start site in the transcription bubble, binds to an initiating
NTP and an extending
NTP (or a short RNA
primer and an extending NTP) complementary to the transcription start site sequence, and catalyzes bond formation to yield an initial RNA product.
In
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...
, RNA polymerase
holoenzyme
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. ...
consists of five subunits: 2 α subunits, 1 β subunit, 1 β' subunit, and 1 ω subunit. In bacteria, there is one general RNA transcription factor known as a
sigma factor. RNA polymerase core enzyme binds to the bacterial general transcription (sigma) factor to form RNA polymerase holoenzyme and then binds to a promoter.
(RNA polymerase is called a holoenzyme when sigma subunit is attached to the core enzyme which is consist of 2 α subunits, 1 β subunit, 1 β' subunit only). Unlike eukaryotes, the initiating nucleotide of nascent bacterial mRNA is not capped with a modified guanine nucleotide. The initiating nucleotide of bacterial transcripts bears a 5′ triphosphate (5′-PPP), which can be used for genome-wide mapping of transcription initiation sites.
In
archaea and
eukaryotes, RNA polymerase contains subunits
homologous to each of the five RNA polymerase subunits in bacteria and also contains additional subunits. In archaea and eukaryotes, the functions of the bacterial general transcription factor sigma are performed by multiple general transcription factors that work together.
In archaea, there are three general transcription factors:
TBP,
TFB, and
TFE. In eukaryotes, in
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 eukaryo ...
-dependent transcription, there are six general transcription factors:
TFIIA
Transcription factor TFIIA is a nuclear protein involved in the RNA polymerase II-dependent transcription of DNA. TFIIA is one of several general (basal) transcription factors ( GTFs) that are required for all transcription events that use RNA ...
,
TFIIB
Transcription factor II B (TFIIB) is a general transcription factor that is involved in the formation of the RNA polymerase II preinitiation complex (PIC) and aids in stimulating transcription initiation. TFIIB is localised to the nucleus and pr ...
(an
ortholog
Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a s ...
of archaeal TFB),
TFIID
Transcription factor II D (TFIID) is one of several general transcription factors that make up the RNA polymerase II preinitiation complex. RNA polymerase II holoenzyme is a form of eukaryotic RNA polymerase II that is recruited to the promoters o ...
(a multisubunit factor in which the key subunit,
TBP, is an
ortholog
Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a s ...
of archaeal TBP),
TFIIE
Transcription factor II E (TFIIE) is one of several general transcription factors that make up the RNA polymerase II preinitiation complex. It is a tetramer of two alpha and two beta chains and interacts with TAF6/TAFII80, ATF7IP, and varicella ...
(an
ortholog
Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a s ...
of archaeal TFE),
TFIIF
Transcription factor II F (TFIIF) is one of several general transcription factors that make up the RNA polymerase II preinitiation complex.
TFIIF is encoded by the , , and genes.
TFIIF binds to RNA polymerase II
RNA polymerase II (RNAP II ...
, and
TFIIH
Transcription factor II Human (transcription factor II H; TFIIH) is an important protein complex, having roles in transcription of various protein-coding genes and DNA nucleotide excision repair (NER) pathways. TFIIH first came to light in 1989 ...
. The TFIID is the first component to bind to DNA due to binding of TBP, while TFIIH is the last component to be recruited. In archaea and eukaryotes, the RNA polymerase-promoter closed complex is usually referred to as the "
preinitiation complex."
Transcription initiation is regulated by additional proteins, known as
activators and
repressors, and, in some cases, associated
coactivator
A coactivator is a type of transcriptional coregulator that binds to an activator (a transcription factor) to increase the rate of transcription of a gene or set of genes. The activator contains a DNA binding domain that binds either to a DNA ...
s or
corepressors, which modulate formation and function of the transcription initiation complex.
Promoter escape
After the first bond is synthesized, the RNA polymerase must escape the promoter. During this time there is a tendency to release the RNA transcript and produce truncated transcripts. This is called
abortive initiation Abortive initiation, also known as abortive transcription, is an early process of genetic transcription in which RNA polymerase binds to a DNA promoter and enters into cycles of synthesis of short mRNA transcripts which are released before the tra ...
, and is common for both eukaryotes and prokaryotes. Abortive initiation continues to occur until an RNA product of a threshold length of approximately 10 nucleotides is synthesized, at which point promoter escape occurs and a transcription elongation complex is formed.
Mechanistically, promoter escape occurs through
DNA scrunching, providing the energy needed to break interactions between RNA polymerase holoenzyme and the promoter.
In bacteria, it was historically thought that the
sigma factor is definitely released after promoter clearance occurs. This theory had been known as the ''obligate release model.'' However, later data showed that upon and following promoter clearance, the sigma factor is released according to a
stochastic model known as the ''stochastic release model''.
In eukaryotes, at an RNA polymerase II-dependent promoter, upon promoter clearance, TFIIH phosphorylates serine 5 on the carboxy terminal domain of RNA polymerase II, leading to the recruitment of capping enzyme (CE). The exact mechanism of how CE induces promoter clearance in eukaryotes is not yet known.
Elongation
![Simple transcription elongation1](https://upload.wikimedia.org/wikipedia/commons/2/21/Simple_transcription_elongation1.svg)
One strand of the DNA, the ''template strand'' (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy (which elongates during the traversal). Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-template) strand and newly formed RNA can also be used as reference points, so transcription can be described as occurring 5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand (except 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 pyrimidi ...
s 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 b ...
s, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate backbone).
mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA), so many mRNA molecules can be rapidly produced from a single copy of a gene. The characteristic elongation rates in prokaryotes and eukaryotes are about 10–100 nts/sec. In eukaryotes, however,
nucleosomes act as major barriers to transcribing polymerases during transcription elongation.
In these organisms, the pausing induced by nucleosomes can be regulated by transcription elongation factors such as TFIIS.
Elongation also involves a proofreading mechanism that can replace incorrectly incorporated bases. In eukaryotes, this may correspond with short pauses during transcription that allow appropriate RNA editing factors to bind. These pauses may be intrinsic to the RNA polymerase or due to chromatin structure.
Termination
Bacteria use two different strategies for transcription termination – Rho-independent termination and Rho-dependent termination. In
Rho-independent transcription termination
Intrinsic, or rho-independent termination, is a process in prokaryotes to signal the end of transcription and release the newly constructed RNA molecule. In prokaryotes such as E. coli, transcription is terminated either by a rho-dependent proc ...
, RNA transcription stops when the newly synthesized RNA molecule forms a G-C-rich
hairpin loop followed by a run of Us. When the hairpin forms, the mechanical stress breaks the weak rU-dA bonds, now filling the DNA–RNA hybrid. This pulls the poly-U transcript out of the active site of the RNA polymerase, terminating transcription. In the "Rho-dependent" type of termination, a protein factor called "
Rho
Rho (uppercase Ρ, lowercase ρ or ; el, ρο or el, ρω, label=none) is the 17th letter of the Greek alphabet. In the system of Greek numerals it has a value of 100. It is derived from Phoenician letter res . Its uppercase form uses the sa ...
" destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.
Transcription termination in eukaryotes is less well understood than in bacteria, but involves cleavage of the new transcript followed by template-independent addition of adenines at its new 3' end, in a process called
polyadenylation.
Role of RNA polymerase in post-transcriptional changes in RNA
![RNA role in the transcription and interaction with other transcription factors](https://upload.wikimedia.org/wikipedia/commons/3/39/RNA_role_in_the_transcription_and_interaction_with_other_transcription_factors_.png)
RNA polymerase plays a very crucial role in all steps including post-transcriptional changes in RNA.
![Ctd role](https://upload.wikimedia.org/wikipedia/commons/3/31/Ctd_role_.png)
As shown in the image in the right it is evident that the CTD (C Terminal Domain) is a tail that changes its shape; this tail will be used as a carrier of splicing, capping and
polyadenylation, as shown in the image on the left.
Inhibitors
Transcription inhibitors can be used as
antibiotics against, for example,
pathogenic bacteria (
antibacterial
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 ...
s) and
fungi
A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, separately from ...
(
antifungals
An antifungal medication, also known as an antimycotic medication, is a pharmaceutical fungicide or fungistatic used to treat and prevent mycosis such as athlete's foot, ringworm, candidiasis (thrush), serious systemic infections such as cryp ...
). An example of such an antibacterial is
rifampicin, which inhibits
bacterial transcription of DNA into mRNA by inhibiting DNA-dependent
RNA polymerase by binding its beta-subunit, while
8-hydroxyquinoline
8-Hydroxyquinoline (also known as oxine) is a chelate, chelating agent which has been used for the Quantitative analysis (chemistry), quantitative determination of metal ions.
In aqueous solution 8-hydroxyquinoline has a pKa value of ca. 9.9 It r ...
is an antifungal transcription inhibitor. The effects of
histone methylation may also work to inhibit the action of transcription. Potent, bioactive natural products like triptolide that inhibit mammalian transcription via inhibition of the XPB subunit of the general transcription factor TFIIH has been recently reported as a glucose conjugate for targeting hypoxic cancer cells with increased glucose transporter production.
Endogenous inhibitors
In vertebrates, 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 ...
the gene becomes inhibited (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 hav ...
or passenger mutations.
However, transcriptional inhibition (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 inhibited 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 Cancer epigenetics, epigenetic mechanisms, such as altered production of MicroRNA#DNA repair and cancer, microRNAs.
In breast cancer, transcriptional repression of BRCA1 may occur more frequently by over-produced microRNA-182 than by hypermethylation of the BRCA1 promoter (see BRCA1#Low expression of BRCA1 in breast and ovarian cancers, Low expression of BRCA1 in breast and ovarian cancers).
Transcription factories
Active transcription units are clustered in the nucleus, in discrete sites called transcription factories or euchromatin. Such sites can be visualized by allowing engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U) and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using fluorescence in situ hybridization or marked by antibodies directed against polymerases. There are ~10,000 factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with only one polymerase, each factory usually contains ~8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a "cloud" around the factor.
History
A molecule that allows the genetic material to be realized as a protein was first hypothesized by François Jacob and Jacques Monod. Severo Ochoa won a Nobel Prize in Physiology or Medicine in 1959 for developing a process for synthesizing RNA ''in vitro'' with polynucleotide phosphorylase, which was useful for cracking the genetic code. RNA synthesis by
RNA polymerase was established ''in vitro'' by several laboratories by 1965; however, the RNA synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate transcription correctly.
In 1972, Walter Fiers became the first person to actually prove the existence of the terminating enzyme.
Roger D. Kornberg won the 2006 Nobel Prize in Chemistry "for his studies of the molecular basis of eukaryotic transcription".
Measuring and detecting
![Transcription label en](https://upload.wikimedia.org/wikipedia/commons/4/43/Transcription_label_en.jpg)
Transcription can be measured and detected in a variety of ways:
* G-Less Cassette transcription assay: measures promoter strength
* Run-off transcription assay: identifies transcription start sites (TSS)
* Nuclear run-on assay: measures the relative abundance of newly formed transcripts
* KAS-seq: measures single-stranded DNA generated by RNA polymerases; can work with 1,000 cells.
* RNase protection assay and ChIP-Chip of RNAP: detect active transcription sites
* RT-PCR: measures the absolute abundance of total or nuclear RNA levels, which may however differ from transcription rates
* DNA microarrays: measures the relative abundance of the global total or nuclear RNA levels; however, these may differ from transcription rates
* In situ hybridization: detects the presence of a transcript
* MS2 tagging: by incorporating RNA Stem-loop, stem loops, such as MS2, into a gene, these become incorporated into newly synthesized RNA. The stem loops can then be detected using a fusion of GFP and the MS2 coat protein, which has a high affinity, sequence-specific interaction with the MS2 stem loops. The recruitment of GFP to the site of transcription is visualized as a single fluorescent spot. This new approach has revealed that transcription occurs in discontinuous bursts, or pulses (see Transcriptional bursting). With the notable exception of in situ techniques, most other methods provide cell population averages, and are not capable of detecting this fundamental property of genes.
* Northern blot: the traditional method, and until the advent of RNA-Seq, the most quantitative
* RNA-Seq: applies next-generation sequencing techniques to sequence whole transcriptomes, which allows the measurement of relative abundance of RNA, as well as the detection of additional variations such as fusion genes, post-transcriptional edits and novel splice sites
* Single-cell RNA-sequencing, Single cell RNA-Seq: amplifies and reads partial transcriptomes from isolated cells, allowing for detailed analyses of RNA in tissues, embryos, and cancers
Reverse transcription
![RetroTranscription](https://upload.wikimedia.org/wikipedia/commons/a/a2/RetroTranscription.jpg)
Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is ''reverse transcribed'' into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase.
In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. The enzyme ribonuclease H then digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure ("cDNA"). The cDNA is integrated into the host cell's genome by the enzyme integrase, which causes the host cell to generate viral proteins that reassemble into new viral particles. In HIV, subsequent to this, the host cell undergoes programmed cell death, or apoptosis of T cells.
However, in other retroviruses, the host cell remains intact as the virus buds out of the cell.
Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes a repeating sequence of DNA, or "junk" DNA. This repeated sequence of DNA is called a telomere and can be thought of as a "cap" for a chromosome. It is important because every time a linear chromosome is duplicated, it is shortened. With this "junk" DNA or "cap" at the ends of chromosomes, the shortening eliminates some of the non-essential, repeated sequence rather than the protein-encoding DNA sequence, that is farther away from the chromosome end.
Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes indefinitely without losing important protein-coding DNA sequence. Activation of telomerase could be part of the process that allows cancer cells to become ''immortal''. The immortalizing factor of cancer via telomere lengthening due to telomerase has been proven to occur in 90% of all carcinogenic tumors ''in vivo'' with the remaining 10% using an alternative telomere maintenance route called ALT or Alternative Lengthening of Telomeres.
See also
* Life
* Cell (biology)
* Cell division
* DBTSS
* gene
* gene regulation
* gene expression
* Epigenetics
* Genome
* Crick's central dogma, in which the product of transcription, mRNA, is Translation (genetics), translated to form polypeptides, and where it is asserted that the reverse processes never occur
* Gene regulation
* Long non-coding RNA
* Missense mRNA
* splicing (genetics), Splicing – process of removing introns from precursor messenger RNA (pre-mRNA) to make messenger RNA (mRNA)
* Transcriptomics
* Translation (biology)
References
External links
Interactive Java simulation of transcription initiation. Fro
Center for Models of Lifeat the Niels Bohr Institute.
From [http://cmol.nbi.dk/ Center for Models of Life] at the Niels Bohr Institute.
Virtual Cell Animation Collection, Introducing Transcription
{{DEFAULTSORT:Transcription (Genetics)
Gene expression
Molecular biology
Cellular processes