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. A 550 kDa complex of 12 subunits, RNAP II is the most studied type of RNA polymerase. A wide range of transcription factors are required for it to bind to its promoters and begin transcription.

Stages of transcription
In the process of transcription (by any polymerase) there are three main stages:
 * 1) Initiation; the construction of the RNA polymerase complex on the gene's promoter with the help of transcription factors.
 * 2) Elongation; the actual transcription of the majority of the gene into a corresponding RNA sequence, highly moderated by several methods.
 * 3) Termination; the cessation of RNA transcription and the disassembly of the RNA polymerase complex.

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
Preinitiation complex (PIC): the construction of the polymerase complex on the promoter. The TATA box is one well-studied example of a promoter element. 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 is located at approximately 25 nucleotides upstream of the Transcription Start Point (TSP). In addition, there are also some weakly conserved features including the B-Recognition Element (BRE), approximately 5 nucleotides upstream of the TATA box.

Order in which the GTFs attach
The following is the order in which the GTFs (general transcription factors) attach:


 * 1) TBP (TATA Binding Protein, see TATA Binding Protein) and an attached complex of TAFs (TBP Associated Factors), collectively known as TFIID (Transcription Factor for polymerase II D), bind at the TATA box.†
 * 2) TFIIA (three subunits) binds TFIID and DNA, stabilizing the first interactions.
 * 3) TFIIB binds between TFIID and the location of Pol II binding in the near future. TFIIB binds partially sequence specifically, with some preference for BRE.
 * 4) TFIIF and Pol II (two subunits, RAP30 and RAP74, showing some similarity to bacterial sigma factors) enter the complex together. TFIIF helps to speed up the polymerization process.
 * 5) TFIIE enters the complex, and helps to open and close the Pol II’s ‘Jaw’ like structure, which enables movement down the DNA strand. TFIIE and TFIIH enter concomitantly.
 * 6) Finally TFIIH binds. 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 dependently) 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, which marks the end of initiation and the start of elongation. 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.
 * 7) Mediator then encases all the transcription factors and the Pol II. Mediator interacts with enhancers, areas very far away (upstream or downstream) that help regulate transcription.

†Occasionally there is no TATA box at the promoter. In this case a TAF will bind sequence specifically, and force the TBP to bind non sequence specifically. TAFs are highly variable, and add a level of control to the initiation.

Initiation Regulation
Initiation is regulated by many mechanisms. These can be separated into two main categories:
 * 1) Protein interference.
 * 2) Chromatin structure inhibition.

Regulation by Protein interference
Protein interference is the process where some signaling protein interacts, either with the promoter or some stage of the partially constructed complex, to prevent further construction of the polymerase complex, so preventing initiation. This is generally 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 structure inhibition is the process where the promoter is hidden by chromatin structure. Chromatin structure is controlled by post-translational modification of the histones involved and leads to gross levels of high or low transcription levels. See: chromatin, histone and nucleosome. For a detailed treatment of a single example see: RNA polymerase control by chromatin structure.

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 that is called the CTD (C-terminal domain). This is the target of kinases 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 Serines and Threonines 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 complementary RNA nucleotides to the template DNA by Watson-Crick base pairing. These RNA nucleotides are ligated and this results in a strand of messenger RNA.

Elongation Regulation
RNA Pol II elongation promoters can be summarised in 3 classes:


 * 1) Drug/sequence-dependent arrest affected factors. Eg. SII (TFIIS) and P-TEFb protein families.
 * 2) Chromatin structure oriented factors. Based on histone post translational modifications - phosphorylation, acetylation, methylation and ubiquination.
 * See: chromatin, histone, and nucleosome
 * 1) 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 kinetics, Henri-Michaelis-Menten kinetics, Michaelis constant, and Lineweaver-Burk diagram

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 chromating 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. For detailed treatment of an example see: RNA polymerase control by chromatin structure.

Termination
Termination is the process of breaking up of the polymerase complex and ending of 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 RNA 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.