RNA Polymerase II Holoenzyme
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RNA polymerase II holoenzyme is a form of
eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
RNA polymerase II that is recruited to the promoters of
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
-coding genes in living cells. It consists of
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a Protein complex, multiprotein complex that Transcription (biology), transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNA pol ...
, a subset of general
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
s, and regulatory proteins known as .


RNA polymerase II

RNA polymerase II (also called RNAP II and Pol II) is an enzyme found in
eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
cells. It catalyzes the transcription of
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
to synthesize precursors of
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
and most snRNA and
microRNA Micro ribonucleic acid (microRNA, miRNA, μRNA) are small, single-stranded, non-coding RNA molecules containing 21–23 nucleotides. Found in plants, animals, and even some viruses, miRNAs are involved in RNA silencing and post-transcr ...
. In humans, RNAP II consists of seventeen protein molecules (gene products encoded by POLR2A-L, where the proteins synthesized from '' POLR2C'', ''
POLR2E DNA-directed RNA polymerases I, II, and III subunit RPABC1 is a protein that in humans is encoded by the ''POLR2E'' gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a se ...
'', and '' POLR2F'' form homodimers).


General transcription factors

General transcription factors (GTFs) or basal transcription factors are
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
s that have been shown to be important in the transcription of class II genes to
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
templates. Many of them are involved in the formation of a preinitiation complex, which, together with
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a Protein complex, multiprotein complex that Transcription (biology), transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNA pol ...
, 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
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
s that is necessary for the transcription of protein-coding
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
s in
eukaryote The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
s and
archaea Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
. The PIC helps position RNA polymerase II over gene
transcription start site Transcription is the process of copying a segment of DNA into RNA for the purpose of gene expression. Some segments of DNA are transcribed into RNA molecules that can encode proteins, called messenger RNA (mRNA). Other segments of DNA are transc ...
s, denatures the DNA, and positions the DNA in the RNA polymerase II
active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...
for transcription. The typical PIC is made up of six general transcription factors: TFIIA ( GTF2A1, GTF2A2), TFIIB ( GTF2B), B-TFIID (
BTAF1 TATA-binding protein-associated factor 172 is a protein that in humans is encoded by the ''BTAF1'' gene. Function Initiation of transcription by RNA polymerase II requires the assistance of TATA box-binding protein (TBP; MIM 600075) and TBP-as ...
, TBP), TFIID (
BTAF1 TATA-binding protein-associated factor 172 is a protein that in humans is encoded by the ''BTAF1'' gene. Function Initiation of transcription by RNA polymerase II requires the assistance of TATA box-binding protein (TBP; MIM 600075) and TBP-as ...
, BTF3, BTF3L4, EDF1, TAF1-15, 16 total), TFIIE, TFIIF, TFIIH and TFIIJ. The construction of the polymerase complex takes place on the
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
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.


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), TBPL1, or TBPL2 can bind the promoter or TATA box. Most
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
s lack a TATA box and use an
initiator element The initiator element (''Inr''), sometimes referred to as initiator motif, is a core promoter that is similar in function to the Pribnow box (in prokaryotes) or the TATA box (in eukaryotes). The ''Inr'' is the simplest functional promoter that i ...
(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 box 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-box 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 The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
domain of TFIIB brings the DNA into proper position for entry into the active site of
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a Protein complex, multiprotein complex that Transcription (biology), transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNA pol ...
. TFIIB binds partially sequence specifically, with some preference for BRE. The TFIID-TFIIA-TFIIB (DAB)-promoter complex subsequently recruits RNA polymerase II and TFIIF. # TFIIF (two subunits, RAP30 and RAP74, showing some similarity to bacterial
sigma factor A sigma factor (σ factor or specificity factor) is a protein needed for initiation of Transcription (biology), transcription in bacteria. It is a bacterial transcription initiation factor that enables specific binding of RNA polymerase (RNAP) to g ...
s) and Pol II 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 Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (''Tm'') is defined as the temperature at which half of the DNA strands are in the random coil ...
at the promoter: it contains a zinc ribbon motif that can bind single-stranded DNA. TFIIE helps to open and close the Pol II’s ''Jaw''-like structure, which enables movement down the DNA strand. #
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
may be wrapped one complete turn around the preinitiation complex and it is TFIIF that helps keep this tight wrapping. In the process, the torsional strain on the DNA may aid in
DNA melting Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (''Tm'') is defined as the temperature at which half of the DNA strands are in the random coil ...
at the promoter, forming the transcription bubble. # TFIIH enters the complex. 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 ( ATP-dependent) to separate the two strands using its
helicase Helicases are a class of enzymes that are vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic double helix, separating the two hybridized ...
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
RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
. Finally it is essential for
Nucleotide Excision Repair Nucleotide excision repair is a DNA repair mechanism. DNA damage occurs constantly because of chemicals (e.g. Intercalation (biochemistry), intercalating agents), radiation and other mutagens. Three excision repair pathways exist to repair single ...
(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 bubble and may be required for transcription if the
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
template is not already denatured or if it is supercoiled. # Mediator then encases all the transcription factors and Pol II. It interacts with enhancers, 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 (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
l initiation. In bacteria, the
sigma factor A sigma factor (σ factor or specificity factor) is a protein needed for initiation of Transcription (biology), transcription in bacteria. It is a bacterial transcription initiation factor that enables specific binding of RNA polymerase (RNAP) to g ...
recognizes and binds to the promoter sequence. In
eukaryote The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
s, the
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
s perform this role.


Mediator complex

Mediator is a multiprotein complex that functions as a transcriptional coactivator. 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 RNA polymerase II (RNAP II and Pol II) is a Protein complex, multiprotein complex that Transcription (biology), transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNA pol ...
holoenzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
, acting as a bridge between this enzyme and
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
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
cap A cap is a flat headgear, usually with a visor. Caps have crowns that fit very close to the head. They made their first appearance as early as 3200 BC. The origin of the word "cap" comes from the Old French word "chapeau" which means "head co ...
ping of the RNA transcript, and attachment to the
spliceosome A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs ( snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to sp ...
for
RNA splicing RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcription (biology), transcript is transformed into a mature messenger RNA (Messenger RNA, mRNA). It works by removing all the introns (non-cod ...
. The CTD typically consists of up to 52 repeats (in humans) 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 In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...
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 The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
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.


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
polyadenylation Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA (mRNA). The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In euka ...
. Ser5 phosphorylation (Ser5PO4) near the 5′ ends of genes depends principally on the kinase activity of TFIIH (Kin28 in
yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...
; CDK7 in
metazoan Animals are multicellular, eukaryotic organisms in the biological kingdom Animalia (). With few exceptions, animals consume organic material, breathe oxygen, have myocytes and are able to move, can reproduce sexually, and grow from a ho ...
s). 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 Cyclin-dependent kinase 9 or CDK9 is a cyclin-dependent kinase associated with P-TEFb. Function The protein encoded by this gene is a member of the cyclin-dependent kinase (CDK) family. CDK family members are highly similar to the gene produ ...
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 An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
that phosphorylate the carboxy-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 In biochemistry, a phosphatase is an enzyme that uses water to cleave a phosphoric acid Ester, monoester into a phosphate ion and an Alcohol (chemistry), alcohol. Because a phosphatase enzyme catalysis, catalyzes the hydrolysis of its Substrate ...
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 (RNAP II and Pol II) is a Protein complex, multiprotein complex that Transcription (biology), transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNA pol ...
. 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 Cyclin-dependent kinase 9 or CDK9 is a cyclin-dependent kinase associated with P-TEFb. Function The protein encoded by this gene is a member of the cyclin-dependent kinase (CDK) family. CDK family members are highly similar to the gene produ ...
kinase In biochemistry, a kinase () is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule don ...
, both of which are involved in the
phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...
and regulation. : ATP + [DNA-directed
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a Protein complex, multiprotein complex that Transcription (biology), transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNA pol ...
] <=> Adenosine diphosphate, ADP + [DNA-directed RNA polymerase II] phosphate : catalyzed by
CDK9 Cyclin-dependent kinase 9 or CDK9 is a cyclin-dependent kinase associated with P-TEFb. Function The protein encoded by this gene is a member of the cyclin-dependent kinase (CDK) family. CDK family members are highly similar to the gene produ ...
EC 2.7.11.23. 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).


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
spliceosome A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs ( snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to sp ...
factors that are part of
RNA splicing RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcription (biology), transcript is transformed into a mature messenger RNA (Messenger RNA, mRNA). It works by removing all the introns (non-cod ...
. 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 Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the Proteinogenic amino acid, 22 α-amino acids incorporated into p ...
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
BRCA1 Breast cancer type 1 susceptibility protein is a protein that in humans is encoded by the ''BRCA1'' () gene. Orthologs are common in other vertebrate species, whereas invertebrate genomes may encode a more distantly related gene. ''BRCA1'' is a ...
-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 'ménage à 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 DNA repair is a collection of processes by which a cell (biology), cell identifies and corrects damage to the DNA molecules that encode its genome. A weakened capacity for DNA repair is a risk factor for the development of cancer. DNA is cons ...
. The
nucleotide excision repair Nucleotide excision repair is a DNA repair mechanism. DNA damage occurs constantly because of chemicals (e.g. Intercalation (biochemistry), intercalating agents), radiation and other mutagens. Three excision repair pathways exist to repair single ...
(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+.


Transcription coactivators

The EDF1 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.


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
archaea Archaea ( ) is a Domain (biology), domain of organisms. Traditionally, Archaea only included its Prokaryote, prokaryotic members, but this has since been found to be paraphyletic, as eukaryotes are known to have evolved from archaea. Even thou ...
domain is similar to transcription in
eukaryote The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
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 () is the physical quantity, quantitative physical property, property that is transferred to a physical body, body or to a physical system, recognizable in the performance of Work (thermodynamics), work and in the form of heat and l ...
-dependent process, consuming
adenosine triphosphate Adenosine triphosphate (ATP) is a nucleoside triphosphate that provides energy to drive and support many processes in living cell (biology), cells, such as muscle contraction, nerve impulse propagation, and chemical synthesis. Found in all known ...
(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 prokaryotes. 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 that Pol II transcribes, this is the polymerase that experiences the most 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 where in 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 is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...
structure inhibition is the process wherein the promoter is hidden by
chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...
structure. Chromatin structure is controlled by post-translational modification of the
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei and in most Archaeal phyla. They act as spools around which DNA winds to create structural units called nucleosomes ...
s involved and leads to gross levels of high or low transcription levels. See:
chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...
,
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei and in most Archaeal phyla. They act as spools around which DNA winds to create structural units called nucleosomes ...
, and
nucleosome A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone, histone proteins and resembles thread wrapped around a bobbin, spool. The nucleosome ...
. 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
kinases In biochemistry, a kinase () is an enzyme that catalysis, catalyzes the transfer of phosphate groups from High-energy phosphate, high-energy, phosphate-donating molecules to specific Substrate (biochemistry), substrates. This process is known as ...
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 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 ...
. The CTD consists of repetitions of an
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
motif, YSPTSPS, of which
Serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − ...
s and
Threonine Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form when dissolved in water), a carboxyl group (which is in the deprotonated −COO− ...
s can be
phosphorylated In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writt ...
. 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 complementary RNA nucleotides to the template DNA by Watson-Crick
base pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
ing. These RNA nucleotides are ligated, resulting in a strand of
messenger RNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...
. 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 three classes: # Drug/sequence-dependent arrest affected factors, e.g., SII (TFIIS) and
P-TEFb The positive transcription elongation factor, P-TEFb, is a multiprotein complex that plays an essential role in the regulation of transcription by RNA polymerase II (Pol II) in eukaryotes. Immediately following initiation Pol II becomes trapped i ...
protein families. # Chromatin structure oriented factors. Based on histone post translational modifications – phosphorylation, acetylation, methylation and ubiquination. #: ''See:
chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...
,
histone In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei and in most Archaeal phyla. They act as spools around which DNA winds to create structural units called nucleosomes ...
, and
nucleosome A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone, histone proteins and resembles thread wrapped around a bobbin, spool. The nucleosome ...
'' # RNA Pol II catalysis improving factors. Improve the Vmax or Km of RNA Pol II, so improving the catalytic quality of the polymerase enzyme. E.g. TFIIF, Elongin and ELL families. #: ''See:
Enzyme kinetics Enzyme kinetics is the study of the rates of enzyme catalysis, enzyme-catalysed chemical reactions. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme' ...
, Henri–Michaelis–Menten kinetics, Michaelis constant, and Lineweaver–Burk plot'' 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 The eukaryotes ( ) constitute the domain of Eukaryota or Eukarya, organisms whose cells have a membrane-bound nucleus. All animals, plants, fungi, seaweeds, and many unicellular organisms are eukaryotes. They constitute a major group of ...
using RNA Pol II, this termination is very variable (up to 2000 bases), relying on post transcriptional modification. 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 yet been demonstrated in
eukaryotes The eukaryotes ( ) constitute the domain of Eukaryota or Eukarya, organisms whose cells have a membrane-bound nucleus. All animals, plants, fungi, seaweeds, and many unicellular organisms are eukaryotes. They constitute a major group of ...
.


Transcription factory

Active RNA Pol II transcription holoenzymes can be clustered in the nucleus, in discrete sites called '' transcription factories''. 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 In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
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 I RNA polymerase 1 (also known as Pol I) is, in higher eukaryotes, the polymerase that only transcribes ribosomal RNA (but not 5S rRNA, which is synthesized by RNA polymerase III), a type of RNA that accounts for over 50% of the total RNA synthesiz ...
*
RNA polymerase III In eukaryote cells, RNA polymerase III (also called Pol III) is a protein that transcribes DNA to synthesize 5S ribosomal RNA, tRNA, and other small RNAs. The genes transcribed by RNA Pol III fall in the category of "housekeeping" genes whose ex ...
*
Post-transcriptional modification Transcriptional modification or co-transcriptional modification is a set of biological processes common to most eukaryotic cells by which an RNA primary transcript is chemically altered following transcription from a gene to produce a mature, f ...
*
Transcription (genetics) Transcription is the process of copying a segment of DNA into RNA for the purpose of gene expression. Some segments of DNA are transcribed into RNA molecules that can encode proteins, called messenger RNA (mRNA). Other segments of DNA are transc ...
*
Eukaryotic transcription Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Un ...


References

*
RNA Polymerase: Components of the Transcription Initiation Machinery
* *


External links



* {{DEFAULTSORT:Rna Polymerase Ii Holoenzyme Enzymes Protein complexes Gene expression