RNA polymerase II

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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.^[1][2] 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 TFIIB-Recognition Element (BRE), approximately 5 nucleotides upstream (BRE^u) and 5 nucleotides downstream (BRE^d) of the TATA box[1].

Order in which the GTFs attach

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

1. TBP (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 (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.
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 and TFIIJ to 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 (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^[3].

†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. Regulation by phosphorylation.

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.

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

3. 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 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 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.

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.

RNA polymerase control by chromatin structure

This is an outline of an example mechanism of yeast cells by which chromatin structure and histone posttranslational modification help regulate and record the transcription of genes by RNA polymerase II.

This pathway gives examples of regulation at these points of transcription:

• Pre-initiation (promotion by Bre1, histone modification)
• Initiation (promotion by TFIIH, Pol II modification AND promotion by COMPASS, histone modification)
• Elongation (promotion by Set2, Histone Modification)

Please note that this refers to various stages of the process as regulatory steps. It has not been proven that they are used for regulation, but is very likely they are.

RNA Pol II elongation promoters can be summarised in 3 classes.

1. Drug/sequence-dependent arrest affected factors (Various interfering proteins).
2. Chromatin structure oriented factors (Histone posttranscriptional modifiers, eg HMTs).
3. RNA Pol II catalysis improving factors (Various interfering proteins and Pol II cofactors, see RNA polymerase II).

Protein Complexes Involved

We are mostly concerned with chromatin structure oriented factors, although there are other factors that become involved in the pathway:

RNA Pol II cofactors:
TFIIH - Phosphorylates C terminal domain (CTD) of the largest RNA Pol II subunit - serine 5. Acts to switch the RNA Pol II into elongation from initiation. Ctk1 - Phosphorylates C terminal domain (CTD) of the largest RNA Pol II subunit - serine 2. Acts in compliment to phosphorylation of serine 5 and is thus seen in middle to late elongation.

Chromatin structure oriented factors:
(HMTs (Histone MethylTransferases)):
COMPASS§† - (COMplex of Proteins ASsociated with Set1) - Methylates lysine 4 of histone H3.
Set2 - Methylates lysine 36 of histone H3.
(interesting irrelevant example: Dot1*‡ - Methylates lysine 79 of histone H3.)

(Other): Bre1 - Ubiquinates (adds ubiquitin to) lysine 123 of histone H2B. Associated with pre-initiation and allowing RNA Pol II binding.

C-terminal domain

Transcription Initiation

The carboxy-terminal domain (CTD) of RNA polymerase II is that portion of the polymerase which is involved in the initiation of DNA transcription. The CTD typically consists of up to 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser ^[4]. 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.

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 have been carried out in which knockout of particular amino acids was achieved in the CTD. 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.

See also

• RNA polymerase I
• RNA polymerase III
• Post-transcriptional modification
• Transcription (genetics)
• Eukaryotic transcription

References

• Lehninger Principles of Biochemistry, 4th edition, David L. Nelson & Michael M. Cox

External links

• More information at Berkeley National Lab

External links

• MeSH RNA+Polymerase+II