RNA polymerase II holoenzyme
Encyclopedia
RNA polymerase II holoenzyme is a form of eukaryotic
RNA polymerase II that is recruited to the promoters of protein
-coding genes in living cells. It consists of RNA polymerase II
, a subset of general transcription factor
s, and regulatory protein
s known as SRB proteins.
cells. It catalyzes the transcription
of DNA
to synthesize precursors of mRNA and most snRNA and microRNA. In humans RNAP II consists of seventeen protein molecules (gene products encoded by POLR2A-L, where the proteins synthesized from 2C-, E-, and F-form homodimers).
transcription factor
s that have been shown to be important in the transcription
of class II gene
s to mRNA templates. Many of them are involved in the formation of a preinitiation complex
, which, together with RNA polymerase II
, bind to and read the single-stranded DNA gene template. The cluster of RNA polymerase II and various transcription factors is known as a basal transcriptional complex (BTC).
s that is necessary for the transcription
of protein-coding gene
s in eukaryote
s and archaea
. The PIC helps position RNA polymerase II over gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site
for transcription.
The typical PIC is made up of six general transcription factors: TFIIA (GTF2A1
, GTF2A2), TFIIB (GTF2B), B-TFIID
(BTAF1
, TBP), TFIID (BTAF1
, BTF3
, BTF3L4, EDF1
, TAF1-15, 16 total), TFIIE, TFIIF, and TFIIH.
The construction of the polymerase complex takes place on the gene
promoter. The TATA box
is one well-studied example of a promoter element that occurs in approximately 10% of genes. It is conserved
in many (though not all) model eukaryotes and is found in a fraction of the promoters in these organisms. The sequence TATA (or variations) is located at approximately 25 nucleotides upstream of the Transcription Start Point (TSP). In addition, there are also some weakly conserved
features including the TFIIB-Recognition Element (BRE), approximately 5 nucleotides upstream (BREu) and 5 nucleotides downstream (BREd) of the TATA box.
The formation of the preinitiation complex (PIC) is analogous to the mechanism seen in bacteria
l initiation. In bacteria, the sigma factor
recognizes and binds to the promoter sequence. In eukaryote
s, the transcription factor
s perform this role.
. The Mediator complex is required for the successful transcription
of nearly all class II gene
promoters in yeast. It works in the same manner in mammals.
The mediator functions as a coactivator and binds to the C-terminal domain (CTD) of RNA polymerase II
holoenzyme, acting as a bridge between this enzyme and transcription factor
s.
ping of the RNA transcript
, and attachment to the spliceosome
for RNA splicing
. The CTD typically consists of up to 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The carboxy-terminal repeat domain (CTD) is essential for life. Cells containing only RNAPII with none or only up to one-third of its repeats are inviable.
The CTD is an extension appended to the C terminus of RPB1, the largest subunit of RNA polymerase II. It serves as a flexible binding scaffold
for numerous nuclear factors, determined by the phosphorylation
patterns on the CTD repeats. Each repeat contains an evolutionary conserved and repeated heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, which is subjected to reversible phosphorylations during each transcription cycle. This domain is inherently unstructured yet evolutionarily conserved, and in eukaryote
s it comprises from 25 to 52 tandem copies of the consensus repeat heptad. As the CTD is frequently not required for general transcription factor
(GTF)-mediated initiation and RNA synthesis, it does not form a part of the catalytic essence of RNAPII, but performs other functions.
.
Ser5 phosphorylation (Ser5PO4) near the 5′ ends of genes depends principally on the kinase activity of TFIIH (Kin28 in yeast
; CDK7 in metazoans). The transcription factor TFIIH is a kinase and will hyperphosphorylate the CTD of RNAP, and in doing so, causes the RNAP complex to move away from the initiation site. Subsequent to the action of TFIIH kinase, Ser2 residues are phosphorylated by CTDK-I in yeast (CDK9
kinase in metazoans). Ctk1 (CDK9) acts in complement to phosphorylation of serine 5 and is, thus, seen in middle to late elongation.
CDK8 and cyclin C (CCNC) are components of the RNA polymerase II holoenzyme that phosphorylate the carboxyl-terminal domain (CTD). CDK8 regulates transcription by targeting the CDK7/cyclin H
subunits of the general transcription initiation factor IIH (TFIIH), thereby providing a link between the mediator and the basal transcription machinery.
The gene CTDP1
encodes a phosphatase
that interacts with the carboxy-terminus of transcription initiation factor TFIIF, a transcription factor that regulates elongation as well as initiation by RNA polymerase II
.
Also involved in the phosphorylation and regulation of the RPB1 CTD is cyclin T1 (CCNT1
). Cyclin T1 tightly associates and forms a complex with CDK9
kinase
, both of which are involved in the phosphorylation
and regulation.
TFIIF and FCP1
cooperate for RNAPII recycling. FCP1, the CTD phosphatase, interacts with RNA polymerase II. Transcription is regulated by the state of phosphorylation of a heptapeptide repeat. The nonphosphorylated form, RNAPIIA, is recruited to the initiation complex, whereas the elongating polymerase is found with RNAPII0. RNAPII cycles during transcription. CTD phosphatase activity is regulated by two GTFs (TFIIF and TFIIB). The large subunit of TFIIF (RAP74) stimulates the CTD phosphatase activity, whereas TFIIB inhibits TFIIF-mediated stimulation. Dephosphorylation of the CTD alters the migration of the largest subunit of RNAPII (RPB1).
The 5'cap of eukaryotic RNA transcripts is important for binding of the mRNA transcript to the ribosome during translation, to the CTD of RNAP, and prevents RNA degradation.
factors that are part of RNA splicing
. These allow for the splicing and removal of introns (in the form of a lariat structure) during RNA transcription.
-associated genome surveillance complex (BASC) associate with RNA polymerase II and play a role in transcription.
The transcription factor TFIIH is involved in transcription initiation and DNA repair. MAT1 (for 'menage a trois-1') is involved in the assembly of the CAK complex. CAK is a multisubunit protein that includes CDK7, cyclin H (CCNH), and MAT1
. CAK is an essential component of the transcription factor TFIIH that is involved in transcription initiation and DNA repair
.
The nucleotide excision repair
(NER) pathway is a mechanism to repair damage to DNA. ERCC2
is involved in transcription-coupled NER and is an integral member of the basal transcription factor BTF2/TFIIH complex. ERCC3 is an ATP-dependent DNA helicase that functions in NER. It also is a subunit of basal transcription factor 2 (TFIIH) and, thus, functions in class II transcription. XPG (ERCC5
) forms a stable complex with TFIIH, which is active in transcription and NER. ERCC6
encodes a DNA-binding protein that is important in transcription-coupled excision repair. ERCC8 interacts with Cockayne syndrome type B (CSB
) protein, with p44 (GTF2H2
), a subunit of the RNA polymerase II transcription factor IIH, and ERCC6. It is involved in transcription-coupled excision repair.
Higher error ratios in transcription by RNA polymerase II are observed in the presence of Mn2+ compared to Mg2+.
gene encodes a protein that acts as a transcriptional coactivator by interconnecting the general transcription factor TATA element-binding protein (TBP) and gene-specific activators.
TFIID and human mediator coactivator (THRAP3
) complexes (mediator complex, plus THRAP3 protein) assemble cooperatively on promoter DNA, from which they become part of the RNAPII holoenzyme.
domain is similar to transcription in eukaryote
s.
Transcription begins with matching of NTPs to the first and second in the DNA sequence. This, like most of the remainder of transcription, is an energy
-dependent process, consuming adenosine triphosphate
(ATP) or other NTP.
Initiation is regulated by many mechanisms. These can be separated into two main categories:
Chromatin
structure inhibition is the process wherein the promoter is hidden by chromatin
structure. Chromatin structure is controlled by post-translational modification of the histone
s involved and leads to gross levels of high or low transcription levels. See: chromatin
, histone
, and nucleosome
.
These methods of control can be combined in a modular method, allowing very high specificity in transcription initiation control.
and phosphatases. The phosphorylation of the CTD is an important regulation mechanism, as this allows attraction and rejection of factors that have a function in the transcription process. The CTD can be considered as a platform for transcription factors.
The CTD consists of repetitions of an amino acid
motif, YSPTSPS, of which Serine
s and Threonine
s can be phosphorylated. The number of these repeats varies; the mammalian protein contains 52, while the yeast protein contains 26. Site-directed-mutagenesis of the yeast protein has found at least 10 repeats are needed for viability. There are many different combinations of phosphorylations possible on these repeats and these can change rapidly during transcription. The regulation of these phosphorylations and the consequences for the association of transcription factors plays a major role in the regulation of transcription.
During the transcription cycle, the CTD of the large subunit of RNAP II is reversibly phosphorylated. RNAP II containing unphosphorylated CTD is recruited to the promoter, whereas the hyperphosphorylated CTD form is involved in active transcription. Phosphorylation occurs at two sites within the heptapeptide repeat, at Serine 5 and Serine 2. Serine 5 phosphorylation is confined to promoter regions and is necessary for the initiation of transcription, whereas Serine 2 phosphorylation is important for mRNA elongation and 3'-end processing.
RNA nucleotides to the template DNA by Watson-Crick base pair
ing. These RNA nucleotides are ligated, resulting in a strand of messenger RNA
.
Unlike DNA replication, 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.
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.
As for initiation, protein interference, seen as the "drug/sequence-dependent arrest affected factors" and "RNA Pol II catalysis improving factors" provide a very rapid response and is used for fine level individual gene control. Elongation downregulation is also possible, in this case usually by blocking polymerase progress or by deactivating the polymerase.
Chromatin structure-oriented factors are more complex than for initiation control. Often the chromatin-altering factor becomes bound to the polymerase complex, altering the histones as they are encountered and providing a semi-permanent 'memory' of previous promotion and transcription.
and Polyadenylation
.
Little regulation occurs at termination, although it has been proposed newly transcribed RNA is held in place if proper termination is inhibited, allowing very fast expression of genes given a stimulus. This has not been demonstrated in eukaryotes as of yet.
. There are ~8,000 such factories in the nucleoplasm of a HeLa cell, but only 100-300 RNAP II foci per nucleus in erythroid cells, as in many other tissue types. The number of transcription factories in tissues is far more restricted than indicated by previous estimates from cultured cells. As an active transcription unit is usually associated with only one Pol II holoenzyme, a polymerase II factory may contain on average ~8 holoenzymes. Colocalization of transcribed genes has not been observed when using cultured fibroblast-like cells. Differentiated or committed tissue types have a limited number of available transcription sites. Estimates show that erythroid cells express at least 4,000 genes, so many genes are obliged to seek out and share the same factory.
The intranuclear position of many genes is correlated with their activity state. During transcription in vivo, distal active genes are dynamically organized into shared nuclear subcompartments and colocalize to the same transcription factory at high frequencies. Movement into or out of these factories results in activation (On) or abatement (Off) of transcription, rather than by recruiting and assembling a transcription complex. Usually, genes migrate to preassembled factories for transcription.
An expressed gene
is preferentially located outside of its chromosome territory, but a closely linked, inactive gene is located inside.
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
RNA polymerase II that is recruited to the promoters of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
-coding genes in living cells. It consists of RNA polymerase II
RNA polymerase II
RNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
, a subset of general transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s, and regulatory protein
Regulation of gene expression
Gene modulation redirects here. For information on therapeutic regulation of gene expression, see therapeutic gene modulation.Regulation of gene expression includes the processes that cells and viruses use to regulate the way that the information in genes is turned into gene products...
s known as SRB proteins.
RNA polymerase II
RNA polymerase II (also called RNAP II and Pol II) is an enzyme found in eukaryoticEukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
cells. It catalyzes the transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
to synthesize precursors of mRNA and most snRNA and microRNA. In humans RNAP II consists of seventeen protein molecules (gene products encoded by POLR2A-L, where the proteins synthesized from 2C-, E-, and F-form homodimers).
General transcription factors
General transcription factors (GTFs) or basal transcription factors are proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s that have been shown to be important in the transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of class II gene
Class II gene
A class II gene is a type of gene that codes for a protein. Class II genes are transcribed by RNAP II.Class II genes have a promoter that often contains a TATA box.Basal transcription of class II genes requires the formation of a preinitiation complex....
s to mRNA templates. Many of them are involved in the formation of a preinitiation complex
Transcription preinitiation complex
The preinitiation complex is a large complex of proteins that is necessary for the transcription of protein-coding genes in eukaryotes...
, which, together with RNA polymerase II
RNA polymerase II
RNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
, bind to and read the single-stranded DNA gene template. The cluster of RNA polymerase II and various transcription factors is known as a basal transcriptional complex (BTC).
Preinitiation complex
The preinitiation complex (PIC) is a large complex of proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s that is necessary for the transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of protein-coding gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s in eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s and archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
. The PIC helps position RNA polymerase II over gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
for transcription.
The typical PIC is made up of six general transcription factors: TFIIA (GTF2A1
GTF2A1
Transcription initiation factor IIA subunit 1 is a protein that in humans is encoded by the GTF2A1 gene.-Interactions:GTF2A1 has been shown to interact with TATA binding protein and TBPL1.-Further reading:...
, GTF2A2), TFIIB (GTF2B), B-TFIID
Transcription Factor II D
RNA polymerase II holoenzyme is a form of eukaryotic RNA polymerase II that is recruited to the promoters of protein-coding genes in living cells. It consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins...
(BTAF1
BTAF1
TATA-binding protein-associated factor 172 is a protein that in humans is encoded by the BTAF1 gene.-Interactions:BTAF1 has been shown to interact with TATA binding protein.-Further reading:...
, TBP), TFIID (BTAF1
BTAF1
TATA-binding protein-associated factor 172 is a protein that in humans is encoded by the BTAF1 gene.-Interactions:BTAF1 has been shown to interact with TATA binding protein.-Further reading:...
, BTF3
BTF3
Transcription factor BTF3 is a protein that in humans is encoded by the BTF3 gene.-Further reading:...
, BTF3L4, EDF1
EDF1
Endothelial differentiation-related factor 1 is a protein that in humans is encoded by the EDF1 gene.-Interactions:EDF1 has been shown to interact with Peroxisome proliferator-activated receptor gamma, TATA binding protein and Liver X receptor alpha....
, TAF1-15, 16 total), TFIIE, TFIIF, and TFIIH.
The construction of the polymerase complex takes place on the gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
promoter. The TATA box
TATA box
The TATA box is a DNA sequence found in the promoter region of genes in archaea and eukaryotes; approximately 24% of human genes contain a TATA box within the core promoter....
is one well-studied example of a promoter element that occurs in approximately 10% of genes. It is conserved
Conservation genetics
Conservation genetics is an interdisciplinary science that aims to apply genetic methods to the conservation and restoration of biodiversity. Researchers involved in conservation genetics come from a variety of fields including population genetics, molecular ecology, biology, evolutionary biology,...
in many (though not all) model eukaryotes and is found in a fraction of the promoters in these organisms. The sequence TATA (or variations) is located at approximately 25 nucleotides upstream of the Transcription Start Point (TSP). In addition, there are also some weakly conserved
Conservation genetics
Conservation genetics is an interdisciplinary science that aims to apply genetic methods to the conservation and restoration of biodiversity. Researchers involved in conservation genetics come from a variety of fields including population genetics, molecular ecology, biology, evolutionary biology,...
features including the TFIIB-Recognition Element (BRE), approximately 5 nucleotides upstream (BREu) and 5 nucleotides downstream (BREd) of the TATA box.
Assembly of the PIC
Although the sequence of steps involved in the assembly of the PIC can vary, in general, they follow step 1, binding to the promoter.- The TATA-binding protein (TBP, a subunit of TFIID), TBPL1TBPL1TATA box-binding protein-like protein 1 is a protein that in humans is encoded by the TBPL1 gene....
, or TBPL2 can bind the promoter or TATA boxTATA boxThe TATA box is a DNA sequence found in the promoter region of genes in archaea and eukaryotes; approximately 24% of human genes contain a TATA box within the core promoter....
. Most geneGeneA gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s lack a TATA boxTATA boxThe TATA box is a DNA sequence found in the promoter region of genes in archaea and eukaryotes; approximately 24% of human genes contain a TATA box within the core promoter....
and use an initiator element (Inr) or downstream core promoter instead. Nevertheless, TBP is always involved and is forced to bind without sequence specificity. TAFs from TFIID can also be involved when the TATA boxTATA boxThe TATA box is a DNA sequence found in the promoter region of genes in archaea and eukaryotes; approximately 24% of human genes contain a TATA box within the core promoter....
is absent. A TFIID TAF will bind sequence specifically, and force the TBP to bind non-sequence specifically, bringing the remaining portions of TFIID to the promoter. - TFIIA interacts with the TBP subunit of TFIID and aids in the binding of TBP to TATA-boxTATA boxThe TATA box is a DNA sequence found in the promoter region of genes in archaea and eukaryotes; approximately 24% of human genes contain a TATA box within the core promoter....
containing promoter DNA. Although TFIIA does not recognize DNA itself, its interactions with TBP allow it to stabilize and facilitate formation of the PIC. - The N-terminal domain of TFIIB brings the DNA into proper position for entry into the active site of RNA polymerase IIRNA polymerase IIRNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
. TFIIB binds partially sequence specifically, with some preference for BRE. The TFIID-TFIIA-TFIIB (DAB)-promoter complex subsequently recruits RNA polymerase II and TFIIF. - TFIIFTranscription Factor II FTranscription factor II F is one of several general transcription factors that make up the RNA polymerase II preinitiation complex.Transcription Factor II F is encoded by the , , and genes. DNA is thought to be wrapped one complete turn around the preinitiation complex and it is TFIIF that...
(two subunits, RAP30GTF2F2General transcription factor IIF subunit 2 is a protein that in humans is encoded by the GTF2F2 gene.-Interactions:GTF2F2 has been shown to interact with POLR2E and HTATSF1.-Further reading:...
and RAP74GTF2F1General transcription factor IIF subunit 1 is a protein that in humans is encoded by the GTF2F1 gene.-Interactions:GTF2F1 has been shown to interact with TAF1, HNRPU, GTF2H4, CTDP1, TATA binding protein, POLR2A, MED21, TAF11, Serum response factor and Transcription Factor II B.-Further reading:...
, showing some similarity to bacterial sigma factorSigma factorA sigma factor is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters. Different sigma factors are activated in response to different environmental conditions...
s) and Pol IIRNA polymerase IIRNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
enter the complex together. TFIIF helps to speed up the polymerization process. - TFIIE joins the growing complex and recruits TFIIH. TFIIE may be involved in DNA melting at the promoter: it contains a zinc ribbonZinc fingerZinc fingers are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families and typically function as interaction modules that bind DNA, RNA, proteins, or small molecules...
motif that can bind single-stranded DNA. TFIIETranscription Factor II ETranscription factor II E is one of several general transcription factors that make up the RNA polymerase II preinitiation complex.Transcription Factor II E is encoded by the GTF2E1 and GTF2E2 genes. TFIIE is thought to be involved in DNA melting at the promoter: it contains a zinc ribbon motif...
helps to open and close the Pol IIRNA polymerase IIRNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
’s Jaw-like structure, which enables movement down the DNA strand. - DNADNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
may be wrapped one complete turn around the preinitiation complexTranscription preinitiation complexThe preinitiation complex is a large complex of proteins that is necessary for the transcription of protein-coding genes in eukaryotes...
and it is TFIIF that helps keep this tight wrapping. In the process, the torsional strain on the DNA may aid in DNA melting at the promoter, forming the transcription bubbleTranscription bubbleA transcription bubble is a molecular structure that occurs during the transcription or replication of DNA when DNA helicase and DNA topoisomerase "unzip" the DNA double strand. DNA polymerase or RNA polymerase may then bind to the exposed DNA and begin synthesizing a new strand of DNA or RNA...
. - TFIIHTranscription Factor II HTranscription factor II H is one of several general transcription factors that make up the RNA polymerase II preinitiation complex. TFIIH consists of ten subunits, 7 of which form the core complex...
and TFIIJ enter the complex together. TFIIH is a large protein complex that contains among others the CDK7/cyclin H kinase complex and a DNA helicase. TFIIH has three functions: It binds specifically to the template strand to ensure that the correct strand of DNA is transcribed and melts or unwinds the DNA (ATPAdenosine triphosphateAdenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
-dependent) to separate the two strands using its helicaseHelicaseHelicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...
activity. It has a kinase activity that phosphorylates the C-terminal domain (CTD) of Pol II at the amino acid serine. This switches the RNA polymerase to start producing RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
. Finally it is essential for Nucleotide Excision RepairNucleotide excision repairNucleotide excision repair is a DNA repair mechanism. DNA constantly requires repair due to damage that can occur to bases from a vast variety of sources including chemicals, radiation and other mutagens...
(NER) of damaged DNA. TFIIH and TFIIE strongly interact with one another. TFIIE affects TFIIH’s catalytic activity. Without TFIIE, TFIIH will not unwind the promoter. - TFIIH helps create the transcription bubbleTranscription bubbleA transcription bubble is a molecular structure that occurs during the transcription or replication of DNA when DNA helicase and DNA topoisomerase "unzip" the DNA double strand. DNA polymerase or RNA polymerase may then bind to the exposed DNA and begin synthesizing a new strand of DNA or RNA...
and may be required for transcription if the DNADNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
template is not already denatured or if it is supercoiledDNA supercoilDNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on the polymer. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to...
. - Mediator then encases all the transcription factors and Pol II. It interacts with enhancerEnhancer (genetics)In genetics, an enhancer is a short region of DNA that can be bound with proteins to enhance transcription levels of genes in a gene cluster...
s, areas very far away (upstream or downstream) that help regulate transcription.
The formation of the preinitiation complex (PIC) is analogous to the mechanism seen in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
l initiation. In bacteria, the sigma factor
Sigma factor
A sigma factor is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters. Different sigma factors are activated in response to different environmental conditions...
recognizes and binds to the promoter sequence. In eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s, the transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s perform this role.
Mediator complex
Mediator is a multiprotein complex that functions as a transcriptional coactivatorCoactivator (genetics)
A coactivator is a protein that increases gene expression by binding to an activator which contains a DNA binding domain. The coactivator is unable to bind DNA by itself....
. The Mediator complex is required for the successful transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of nearly all class II gene
Class II gene
A class II gene is a type of gene that codes for a protein. Class II genes are transcribed by RNAP II.Class II genes have a promoter that often contains a TATA box.Basal transcription of class II genes requires the formation of a preinitiation complex....
promoters in yeast. It works in the same manner in mammals.
The mediator functions as a coactivator and binds to the C-terminal domain (CTD) of RNA polymerase II
RNA polymerase II
RNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
holoenzyme, acting as a bridge between this enzyme and transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s.
C-terminal domain (CTD)
The carboxy-terminal domain (CTD) of RNA polymerase II is that portion of the polymerase that is involved in the initiation of DNA transcription, the cap5' cap
The 5' cap is a specially altered nucleotide on the 5' end of precursor messenger RNA and some other primary RNA transcripts as found in eukaryotes. The process of 5' capping is vital to creating mature messenger RNA, which is then able to undergo translation...
ping of the RNA transcript
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
, and attachment to the spliceosome
Spliceosome
A spliceosome is a complex of snRNA and protein subunits that removes introns from a transcribed pre-mRNA segment. This process is generally referred to as splicing.-Composition:...
for RNA splicing
RNA splicing
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...
. The CTD typically consists of up to 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The carboxy-terminal repeat domain (CTD) is essential for life. Cells containing only RNAPII with none or only up to one-third of its repeats are inviable.
The CTD is an extension appended to the C terminus of RPB1, the largest subunit of RNA polymerase II. It serves as a flexible binding scaffold
Scaffold protein
In biology, scaffold proteins are crucial regulators of many key signaling pathways. Although scaffolds are not strictly defined in function, they are known to interact and/or bind with multiple members of a signaling pathway, tethering them into complexes...
for numerous nuclear factors, determined by the phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
patterns on the CTD repeats. Each repeat contains an evolutionary conserved and repeated heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, which is subjected to reversible phosphorylations during each transcription cycle. This domain is inherently unstructured yet evolutionarily conserved, and in eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s it comprises from 25 to 52 tandem copies of the consensus repeat heptad. As the CTD is frequently not required for general transcription factor
General transcription factor
General transcription factors or basal transcription factors are protein transcription factors that have been shown to be important in the transcription of class II genes to mRNA templates...
(GTF)-mediated initiation and RNA synthesis, it does not form a part of the catalytic essence of RNAPII, but performs other functions.
CTD phosphorylation
RNAPII can exist in two forms: RNAPII0, with a highly phosphorylated CTD, and RNAPIIA, with a nonphosphorylated CTD. Phosphorylation occurs principally on Ser2 and Ser5 of the repeats, although these positions are not equivalent. The phosphorylation state changes as RNAPII progresses through the transcription cycle: The initiating RNAPII is form IIA, and the elongating enzyme is form II0. While RNAPII0 does consist of RNAPs with hyperphosphorylated CTDs, the pattern of phosphorylation on individual CTDs can vary due to differential phosphorylation of Ser2 versus Ser5 residues and/or to differential phosphorylation of repeats along the length of the CTD. The PCTD (phosphoCTD of an RNAPII0) physically links pre-mRNA processing to transcription by tethering processing factors to elongating RNAPII, e.g., 5′-end capping, 3′-end cleavage, and polyadenylationPolyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
.
Ser5 phosphorylation (Ser5PO4) near the 5′ ends of genes depends principally on the kinase activity of TFIIH (Kin28 in yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
; CDK7 in metazoans). The transcription factor TFIIH is a kinase and will hyperphosphorylate the CTD of RNAP, and in doing so, causes the RNAP complex to move away from the initiation site. Subsequent to the action of TFIIH kinase, Ser2 residues are phosphorylated by CTDK-I in yeast (CDK9
CDK9
CDK9 or cyclin-dependent kinase 9 is a cyclin-dependent kinase associated with P-TEFb.CDK9 is also known to associate with other proteins such as TRAF2, and be involved in differentiation of skeletal muscle...
kinase in metazoans). Ctk1 (CDK9) acts in complement to phosphorylation of serine 5 and is, thus, seen in middle to late elongation.
CDK8 and cyclin C (CCNC) are components of the RNA polymerase II holoenzyme that phosphorylate the carboxyl-terminal domain (CTD). CDK8 regulates transcription by targeting the CDK7/cyclin H
Cyclin H
Cyclin-H is a protein that in humans is encoded by the CCNH gene.-Interactions:Cyclin H has been shown to interact with P53, Cyclin-dependent kinase 7 and MNAT1.-Further reading:...
subunits of the general transcription initiation factor IIH (TFIIH), thereby providing a link between the mediator and the basal transcription machinery.
The gene CTDP1
CTDP1
RNA polymerase II subunit A C-terminal domain phosphatase is an enzyme that in humans is encoded by the CTDP1 gene.-Interactions:CTDP1 has been shown to interact with WD repeat-containing protein 77, GTF2F1 and POLR2A.-Further reading:...
encodes a phosphatase
Phosphatase
A phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group . This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their...
that interacts with the carboxy-terminus of transcription initiation factor TFIIF, a transcription factor that regulates elongation as well as initiation by RNA polymerase II
RNA polymerase II
RNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
.
Also involved in the phosphorylation and regulation of the RPB1 CTD is cyclin T1 (CCNT1
CCNT1
Cyclin-T1 is a protein that in humans is encoded by the CCNT1 gene.-Interactions:Cyclin T1 has been shown to interact with HEXIM1, Myc, Aryl hydrocarbon receptor, Granulin, NUFIP1, CDK9 and Promyelocytic leukemia protein.-Further reading:...
). Cyclin T1 tightly associates and forms a complex with CDK9
CDK9
CDK9 or cyclin-dependent kinase 9 is a cyclin-dependent kinase associated with P-TEFb.CDK9 is also known to associate with other proteins such as TRAF2, and be involved in differentiation of skeletal muscle...
kinase
Kinase
In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
, both of which are involved in the phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
and regulation.
- ATPAdenosine triphosphateAdenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
+ [DNA-directed RNA polymerase IIRNA polymerase IIRNA polymerase II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase...
] <=> ADPAdenosine diphosphateAdenosine diphosphate, abbreviated ADP, is a nucleoside diphosphate. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine....
+ [DNA-directed RNA polymerase II] phosphate : catalyzed by CDK9CDK9CDK9 or cyclin-dependent kinase 9 is a cyclin-dependent kinase associated with P-TEFb.CDK9 is also known to associate with other proteins such as TRAF2, and be involved in differentiation of skeletal muscle...
EC 2.7.11.23.
TFIIF and FCP1
CTDP1
RNA polymerase II subunit A C-terminal domain phosphatase is an enzyme that in humans is encoded by the CTDP1 gene.-Interactions:CTDP1 has been shown to interact with WD repeat-containing protein 77, GTF2F1 and POLR2A.-Further reading:...
cooperate for RNAPII recycling. FCP1, the CTD phosphatase, interacts with RNA polymerase II. Transcription is regulated by the state of phosphorylation of a heptapeptide repeat. The nonphosphorylated form, RNAPIIA, is recruited to the initiation complex, whereas the elongating polymerase is found with RNAPII0. RNAPII cycles during transcription. CTD phosphatase activity is regulated by two GTFs (TFIIF and TFIIB). The large subunit of TFIIF (RAP74) stimulates the CTD phosphatase activity, whereas TFIIB inhibits TFIIF-mediated stimulation. Dephosphorylation of the CTD alters the migration of the largest subunit of RNAPII (RPB1).
5' Capping
The carboxy-terminal domain is also the binding site of the cap-synthesizing and cap-binding complex. In eukaryotes, after transcription of the 5' end of an RNA transcript, the cap-synthesizing complex on the CTD will remove the gamma-phosphate from the 5'-phosphate and attach a GMP, forming a 5',5'-triphosphate linkage. The synthesizing complex falls off and the cap then binds to the cap-binding complex (CBC), which is bound to the CTD.The 5'cap of eukaryotic RNA transcripts is important for binding of the mRNA transcript to the ribosome during translation, to the CTD of RNAP, and prevents RNA degradation.
Spliceosome
The carboxy-terminal domain is also the binding site for spliceosomeSpliceosome
A spliceosome is a complex of snRNA and protein subunits that removes introns from a transcribed pre-mRNA segment. This process is generally referred to as splicing.-Composition:...
factors that are part of RNA splicing
RNA splicing
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...
. These allow for the splicing and removal of introns (in the form of a lariat structure) during RNA transcription.
Mutation in the CTD
Major studies in which knockout of particular amino acids was achieved in the CTD have been carried out. The results indicate that RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes.Genome surveillance complex
Several protein members of the BRCA1BRCA1
BRCA1 is a human caretaker gene that produces a protein called breast cancer type 1 susceptibility protein, responsible for repairing DNA. The first evidence for the existence of the gene was provided by the King laboratory at UC Berkeley in 1990...
-associated genome surveillance complex (BASC) associate with RNA polymerase II and play a role in transcription.
The transcription factor TFIIH is involved in transcription initiation and DNA repair. MAT1 (for 'menage a trois-1') is involved in the assembly of the CAK complex. CAK is a multisubunit protein that includes CDK7, cyclin H (CCNH), and MAT1
MNAT1
CDK-activating kinase assembly factor MAT1 is an enzyme that in humans is encoded by the MNAT1 gene.-Interactions:MNAT1 has been shown to interact with POU2F1, MTA1, Cyclin H, P53, Estrogen receptor alpha, MCM7 and Cyclin-dependent kinase 7....
. CAK is an essential component of the transcription factor TFIIH that is involved in transcription initiation and DNA repair
DNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
.
The nucleotide excision repair
Nucleotide excision repair
Nucleotide excision repair is a DNA repair mechanism. DNA constantly requires repair due to damage that can occur to bases from a vast variety of sources including chemicals, radiation and other mutagens...
(NER) pathway is a mechanism to repair damage to DNA. ERCC2
ERCC2
ERCC2, or XPD is a protein involved in transcription-coupled nucleotide excision repair.The XPD gene encodes for a 2.3-kb mRNA containing 22 exons and 21 introns. The XPD protein is a 760 amino acids polypeptide with a size of 87kDa...
is involved in transcription-coupled NER and is an integral member of the basal transcription factor BTF2/TFIIH complex. ERCC3 is an ATP-dependent DNA helicase that functions in NER. It also is a subunit of basal transcription factor 2 (TFIIH) and, thus, functions in class II transcription. XPG (ERCC5
ERCC5
DNA repair protein complementing XP-G cells is a protein that in humans is encoded by the ERCC5 gene.-External links:* -Further reading:...
) forms a stable complex with TFIIH, which is active in transcription and NER. ERCC6
ERCC6
DNA excision repair protein ERCC-6 is a protein that in humans is encoded by the ERCC6 gene.-External links:*...
encodes a DNA-binding protein that is important in transcription-coupled excision repair. ERCC8 interacts with Cockayne syndrome type B (CSB
CSB
CSB or csb can refer to:*California Shuttle Bus a passenger service between the San Francisco Bay Area and the Los Angeles area.* Canada Savings Bond* Canadian Society for Biomechanics...
) protein, with p44 (GTF2H2
GTF2H2
General transcription factor IIH subunit 2 is a protein that in humans is encoded by the GTF2H2 gene.-Interactions:GTF2H2 has been shown to interact with GTF2H5, XPB and ERCC2.-Further reading:...
), a subunit of the RNA polymerase II transcription factor IIH, and ERCC6. It is involved in transcription-coupled excision repair.
Higher error ratios in transcription by RNA polymerase II are observed in the presence of Mn2+ compared to Mg2+.
Transcription coactivators
The EDF1EDF1
Endothelial differentiation-related factor 1 is a protein that in humans is encoded by the EDF1 gene.-Interactions:EDF1 has been shown to interact with Peroxisome proliferator-activated receptor gamma, TATA binding protein and Liver X receptor alpha....
gene encodes a protein that acts as a transcriptional coactivator by interconnecting the general transcription factor TATA element-binding protein (TBP) and gene-specific activators.
TFIID and human mediator coactivator (THRAP3
THRAP3
Thyroid hormone receptor-associated protein 3 is a protein that in humans is encoded by the THRAP3 gene.-Further reading:...
) complexes (mediator complex, plus THRAP3 protein) assemble cooperatively on promoter DNA, from which they become part of the RNAPII holoenzyme.
Transcription initiation
The completed assembly of the holoenzyme with transcription factors and RNA polymerase II bound to the promoter forms the eukaryotic transcription initiation complex. Transcription in the archaeaArchaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
domain is similar to transcription in eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s.
Transcription begins with matching of NTPs to the first and second in the DNA sequence. This, like most of the remainder of transcription, is an energy
Energy
In physics, energy is an indirectly observed quantity. It is often understood as the ability a physical system has to do work on other physical systems...
-dependent process, consuming adenosine triphosphate
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
(ATP) or other NTP.
Promoter clearance
After the first bond is synthesized, the RNA polymerase must clear the promoter. During this time, there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is common for both eukaryotes and prokaroytes. Abortive initiation continues to occur until the σ factor rearranges, resulting in the transcription elongation complex (which gives a 35 bp-moving footprint). The σ factor is released before 80 nucleotides of mRNA are synthesized. Once the transcript reaches approximately 23 nucleotides, it no longer slips and elongation can occur.Initiation Regulation
Due to the range of genes, Pol II transcribes this is the polymerase that experiences greatest regulation, by a range of factors, at each stage of transcription. It is also one of the most complex in terms of polymerase cofactors involved.Initiation is regulated by many mechanisms. These can be separated into two main categories:
- Protein interference.
- Regulation by phosphorylation.
Regulation by Protein interference
Protein interference is the process wherein some signaling protein interacts, either with the promoter or with some stage of the partially constructed complex, to prevent further construction of the polymerase complex, so preventing initiation. In general, this is a very rapid response and is used for fine level, individual gene control and for 'cascade' processes for a group of genes useful under a specific conditions (for example, DNA repair genes or heat shock genes).Chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
structure inhibition is the process wherein the promoter is hidden by chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
structure. Chromatin structure is controlled by post-translational modification of the histone
Histone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
s involved and leads to gross levels of high or low transcription levels. See: chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
, histone
Histone
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
, and nucleosome
Nucleosome
Nucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool....
.
These methods of control can be combined in a modular method, allowing very high specificity in transcription initiation control.
Regulation by Phosphorylation
The largest subunit of Pol II (Rpb1) has a domain at its C-terminus called the CTD (C-terminal domain). This is the target of kinasesProtein kinase
A protein kinase is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them . Phosphorylation usually results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins...
and phosphatases. The phosphorylation of the CTD is an important regulation mechanism, as this allows attraction and rejection of factors that have a function in the transcription process. The CTD can be considered as a platform for transcription factors.
The CTD consists of repetitions of an amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
motif, YSPTSPS, of which Serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
s and Threonine
Threonine
Threonine is an α-amino acid with the chemical formula HO2CCHCHCH3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar...
s can be phosphorylated. The number of these repeats varies; the mammalian protein contains 52, while the yeast protein contains 26. Site-directed-mutagenesis of the yeast protein has found at least 10 repeats are needed for viability. There are many different combinations of phosphorylations possible on these repeats and these can change rapidly during transcription. The regulation of these phosphorylations and the consequences for the association of transcription factors plays a major role in the regulation of transcription.
During the transcription cycle, the CTD of the large subunit of RNAP II is reversibly phosphorylated. RNAP II containing unphosphorylated CTD is recruited to the promoter, whereas the hyperphosphorylated CTD form is involved in active transcription. Phosphorylation occurs at two sites within the heptapeptide repeat, at Serine 5 and Serine 2. Serine 5 phosphorylation is confined to promoter regions and is necessary for the initiation of transcription, whereas Serine 2 phosphorylation is important for mRNA elongation and 3'-end processing.
Elongation
The process of elongation is the synthesis of a copy of the DNA into messenger RNA. RNA Pol II matches complementaryComplementarity (molecular biology)
In molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA, as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds...
RNA nucleotides to the template DNA by Watson-Crick base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
ing. These RNA nucleotides are ligated, resulting in a strand of messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
.
Unlike DNA replication, 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.
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.
Elongation Regulation
RNA Pol II elongation promoters can be summarised in 3 classes:- Drug/sequence-dependent arrest affected factors, e.g., SII (TFIIS) and P-TEFbP-TEFbPositive Transcription Elongation Factor b is the key factor regulating eukaryotic mRNA transcription at the level of elongation.It is a cyclin dependent kinase, composed of Cdk9 and in humans one of three cyclins, T1, T2, or K....
protein families. - Chromatin structure oriented factors. Based on histone post translational modifications - phosphorylation, acetylation, methylation and ubiquination.
- See: chromatinChromatinChromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
, histoneHistoneIn biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation...
, and nucleosomeNucleosomeNucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool....
- See: chromatin
- RNA Pol II catalysis improving factors. Improve the Vmax or Km of RNA Pol II, so improving the catalytic quality of the polymerase enzyme. Eg. TFIIF, Elongin and ELL families.
- See: Enzyme kineticsEnzyme kineticsEnzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction investigated...
, Henri-Michaelis-Menten kinetics, Michaelis constant, and Lineweaver–Burk plot
- See: Enzyme kinetics
As for initiation, protein interference, seen as the "drug/sequence-dependent arrest affected factors" and "RNA Pol II catalysis improving factors" provide a very rapid response and is used for fine level individual gene control. Elongation downregulation is also possible, in this case usually by blocking polymerase progress or by deactivating the polymerase.
Chromatin structure-oriented factors are more complex than for initiation control. Often the chromatin-altering factor becomes bound to the polymerase complex, altering the histones as they are encountered and providing a semi-permanent 'memory' of previous promotion and transcription.
Termination
Termination is the process of breaking up the polymerase complex and ending the RNA strand. In eukaryotes using RNA Pol II, this termination is very variable (up to 2000 bases), relying on post transcriptional modification. See: Messenger RNAMessenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
and Polyadenylation
Polyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
.
Little regulation occurs at termination, although it has been proposed newly transcribed RNA is held in place if proper termination is inhibited, allowing very fast expression of genes given a stimulus. This has not been demonstrated in eukaryotes as of yet.
Transcription factory
Active RNA Pol II transcription holoenzymes can be clustered in the nucleus, in discrete sites called transcription factoriesTranscription factories
In genetics, a transcription factory is an active gene transcription unit that is clustered in a discrete site within the eukaryotic nucleus. Such sites can be visualized by allowing engaged polymerases to extend their transcripts with tagged precursors and immuno-labeling the tagged nascent RNA...
. There are ~8,000 such factories in the nucleoplasm of a HeLa cell, but only 100-300 RNAP II foci per nucleus in erythroid cells, as in many other tissue types. The number of transcription factories in tissues is far more restricted than indicated by previous estimates from cultured cells. As an active transcription unit is usually associated with only one Pol II holoenzyme, a polymerase II factory may contain on average ~8 holoenzymes. Colocalization of transcribed genes has not been observed when using cultured fibroblast-like cells. Differentiated or committed tissue types have a limited number of available transcription sites. Estimates show that erythroid cells express at least 4,000 genes, so many genes are obliged to seek out and share the same factory.
The intranuclear position of many genes is correlated with their activity state. During transcription in vivo, distal active genes are dynamically organized into shared nuclear subcompartments and colocalize to the same transcription factory at high frequencies. Movement into or out of these factories results in activation (On) or abatement (Off) of transcription, rather than by recruiting and assembling a transcription complex. Usually, genes migrate to preassembled factories for transcription.
An expressed gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
is preferentially located outside of its chromosome territory, but a closely linked, inactive gene is located inside.
Holoenzyme stability
RNA polymerase II holoenzyme stability determines the number of base pairs that can be transcribed before the holoenzyme loses its ability to transcribe. The length of the CTD is essential for RNA polymerase II stability. RNA polymerase II stability has been shown to be regulated by post-translation proline hydroxylation. The von Hippel–Lindau tumor suppressor protein (pVHL, human GeneID: 7428) complex binds the hyperphosphorylated large subunit of the RNA polymerase II complex, in a proline hydroxylation- and CTD phosphorylation-dependent manner, targeting it for ubiquitination.See also
- RNA polymerase IRNA polymerase IRNA polymerase I is, in eukaryotes, the enzyme that only transcribes ribosomal RNA , a type of RNA that accounts for over 50% of the total RNA synthesized in a cell....
- RNA polymerase IIIRNA polymerase IIIRNA polymerase III 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 expression is required in all cell types and most environmental conditions...
- Post-transcriptional modificationPost-transcriptional modificationPost-transcriptional modification is a process in cell biology by which, in eukaryotic cells, primary transcript RNA is converted into mature RNA. A notable example is the conversion of precursor messenger RNA into mature messenger RNA , which includes splicing and occurs prior to protein synthesis...
- Transcription (genetics)Transcription (genetics)Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
- Eukaryotic transcriptionEukaryotic transcriptionEukaryotic transcription is more complex than prokaryotic transcription. For instance, in eukaryotes the genetic material , and therefore transcription, is primarily localized to the nucleus, where it is separated from the cytoplasm by the nuclear membrane...