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 .
RNA polymerase II
RNA polymerase II (also called RNAP II and Pol II) is an enzyme found in eukaryotic 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 POLR2C, POLR2E, and POLR2F form homodimers).
General transcription factors
General transcription factors (GTFs) 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. 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).
Preinitiation complex
The preinitiation complex (PIC) is a large complex of proteins that is necessary for the transcription of protein-coding genes in eukaryotes 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, TFIIH and TFIIJ.
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.
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 genes lack a TATA box 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 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 domain of TFIIB brings the DNA into proper position for entry into the active site of RNA polymerase II. 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 factors) 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 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 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 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 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. Finally it is essential for Nucleotide Excision Repair (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 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 bacterial initiation. In bacteria, the sigma factor recognizes and binds to the promoter sequence. In eukaryotes, the transcription factors 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 holoenzyme, acting as a bridge between this enzyme and transcription factors.
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 capping of the RNA transcript, and attachment to the spliceosome for RNA splicing. 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 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 eukaryotes 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.
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 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 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.
ATP + [DNA-directed RNA polymerase II] <=> ADP + [DNA-directed RNA polymerase II] phosphate : catalyzed by CDK9 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 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.
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 BRCA1-associated genome surveillance complex (BASC) associate with RNA polymerase II and play a role in transcription.