# Structural Biochemistry/RNA Polymerase II

Although it has been a common belief that regulation of transcription takes place via regions adjacent to the coding region of the gene, mostly by promoters and enhancers, and that polymerase acts as machine that quickly “reads the gene”, recent evidence suggests that there is much more to the process than just that.
Transcript elongation is extremely complex and highly regulated and the process is significant as it affects both the organization and the integrity of the genome. This will explore some of the intricacies of transcript elongation by RNA Polymerase II, that has been overlooked for some time.

# RNA Polymerase II Transcription Cycle

RNA Polymerase II transcription cycle can be classified into several distinct steps:
* RNAPII is recruited to the promoter of a gene where it forms a pre-initiation complex with the general transcription factors
* At this point initiation ensues and the promoter is left behind in the process called “promoter clearance”
* RNAPII enters processive transcript elongation and the gene is fully transcribed
* Then, transcriptional termination occurs, and results in the release and recycling of the RNAPII

# Overview of Challenges to RNA Polymerase II

Challenges that face RNAPII:
* Polymerase needs to escape the promoter
* The production of pre-mRNA transcript needs to be tightly coupled to RNA biogenesis. This includes RNA capping, splicing, transcript, cleavage, and polyadenylation.
* RNAPII must navigate past nucleosomes and other obstacles (like DNA damage) because transcript elongation occurs in the chromatin
* Transcription is affected by DNA metabolic processes: DNA repair, recombination, and replication.
* Transcript elongation in highly transcribed genes is carried out by several polymerase molecules at the same time, so gene “traffic” must be regulated.

# The Brownian Ratchet Mechanism

This mechanism is what governs transcript elongation. It involves the forward translocation states being stabilized by the binding and hydrolysis of the correct incoming nucleotide. The problem with this mechanism is that although it moves forward rapidly, it can also move backwards. So even though there are newly formed phosphodiester bonds, the enzyme can backtrack for one or several nucleotides so that the newly formed 3’ terminus comes out of alignment with the active site. Brownian motion can bring RNA back in alignment with the active site. General elongation factors affect multiple equilibria between different enzyme states, which is what helps drive the reaction towards forward translocation.

# Transcription Fidelity

Transcription fidelity is essential, as the correct insertion of nucleotides into the RNA transcript during elongation is highly important for accuracy of gene expression.
There are a few key structures of the RNA polymerase II that ensures fidelity:
* Trigger loop
* Rpb9 (subunit on RNAPII)
* Transcription factors, especially transcription factors known as TFIIS
* RNA Polymerase II itself

## The Trigger Loop

The trigger loop is located beneath the active site and is involved in multiple interactions with the incoming nucleotide. The trigger loop plays a key role in fidelity, mismatched nucleotides in the active site do not correctly align with the loop and therefore result in a large reduction of the rate of phosphodiester bond formation. So the trigger loop mediates phosphodiester bond formation in this manner. Trigger loop discriminates against dNTPs and interacts with rNTPs bases and with the 2'OH.

## Rpb9

Another important structure on the RNAPII is the Rpb9 subunit, which was first discovered by scientists studying yeast stains. Rpb9 delays closure of the trigger loop on the incoming nucleotide, which helps to ensure transcription fidelity by allowing more time for mistakes to be fixed.

## RNA Polymerase II and TFIIS

RNAPII itself plays a key role in its own transcription fidelity, as this polymerase can move forwards and backwards upon the molecule it is transcribing, and can therefore correct any mistakes through transcript cleavage of those erroneously incorporated nucleotides. Transcript cleavage is greatly enhanced by transcription factors known as TFIIS. Recent studies have also suggested that TFIIS also play a role in the process of fidelity as they are tightly coupled to the function of Rpb9, which leads us to believe that Rpb9 might be involved in RNAPII fidelity before and after nucleotide addition by affecting the function of the trigger loop and by mediating TFIIS function.

# Transcript Elongation Through Chromatin

Transcript elongation occurs on a chromatin template, chromatin is an extremely repressive template to the process of transcription. Thus, certain mechanisms must be utilized to make it a more friendly place for transcription to occur. This involves temporary displacement and modification/dissasembly of nucleosomes. This can occur through a few different mechanisms, and requires;
* Histone chaperones
* Chromatin remodeling factors
* Histone modifying enzymes
These same factors also aid in the resetting of the chromatin structure after RNAPII has transcribed.

## Nucleosome Assembly

First, the H3-H4 dimers are acetylated by HAT1-RbAp48 complex. Most of the H3 and H4 proteins are then passed on to Asf1, CAF-1, yRtt106, and HIRA to mediate chromatin assembly. The choice between CAF-1, yRtt106, and HIRA depends on the variant of H3. For example, H3.1 targets CAF-1 while H3.3 targets HIRA. CAF-1 and yRtt106 associate with DNA synthesis and HIRA occurs outside of DNA synthesis. Histone hand-off to different histone chaperones depend on the specific acetylation mark H3 K56Ac. H3 K56Ac’s role in yeast is to drive chromatin assembly during DNA repair and replication. However, since H3 K56Ac is harder to detect in humans, it is only speculated that it will do the same task. With H3 K56Ac there is a higher chance that Asf1 will transfer histones to CAF-1 or Rtt106. The choice of the DNA synthesis-dependent pathway or the synthesis-independent pathway depends on the physical interactions between the chaperones. The final chaperone in the hand-off will place the histones on DNA. There are mechanisms for which location of the DNA to place the histone and also mechanisms for increasing the concentration of the histone.

## Nucleosome Dissasembly

One of the mechanisms used is nucleosome disassembly in front of elongating RNAPII; certain experiments prove this to be a mechanism. One such experiment measuring histone density at the yeast GAL genes, showed that gene activation causes loss of nucleosomes at the promoter and also within the coding region. This loss of histone density is caused by elongating RNAPII . Genome-wide analysis of nucleosome occupancy in yeast revealed that transcription rate and histone density are inversely related, further proof that nucleosomes are disassembled during transcription. Another mechanism is the displacement of all core histones during transcription. There are different types of dimers however. H2A/H2B dimers are localized on the exterior of the nucleosome, and have fewer protein-DNA contacts. These dimers are rapidly exchanged in response to transcription factors. Conversely, histones H3 and H4 are much less mobile and their turnover rate is quite independent of the H2A/H2B dimer. So, although all core histones are displaced during this process, histones H2A/H2B are more readily moved while H3/H4 are not.
Just as the nucleosome assembly process occurred in a stepwise manner with histone chaperones, the nucleosome disassembly process is a stepwise process in the reverse direction. The most significant difference between nucleosome assembly and dissambly is that nucleosome disassembly requires energy. The energy is needed to break the histone-DNA bond so that histone chaperones can bind to the histone and move it away from the DNA. The factors to take into account before is undergoing disassembly is the post-translational histone modifications and histone chaperone availability. It is about equilibrium. If only H2A-H2B’s equilibrium is leaning towards removal from DNA there would only be histone exchange. However, if H3-H4’s equilibrium is leaning towards removal from DNA there would be nucleosome disassembly. H3 K56Ac in promoter region is a common example in nucleosome disassembly because it shifts H3-H4’s equilibrium towards removal from DNA. The equilibrium is all affected by interactions that include histone-histone chaperone interaction, histone-DNA interaction, or histone-histone interactions.

### Histone Chaperones

Histone chaperones are histone-binding proteins involved in intracellular histone dynamics, recent evidence revealed that one histone chaperone known as FACT has a huge role in transcription elongation. FACT (facilitates chromatin transcription) is a histone chaperone that facilitates elongation by destabilizing nucleosome structure so that H2A/H2B dimer can be removed during the passage of RNAPII. Spt6 is a H3 and H4 histone chaperone that maintains chromatin structure, promoting restoration of normal chromatin structure in the wake of RNAPII transcript elongation. Histone chaperones in general are involved in both removing and re-depositing histones during transcript elongation.
Histone chaperones as proteins not only pack histone and DNA into the nucleosome structure but also dissembles the structure. There are certain ways histones and DNA fold together in the nucleosome and histone chaperones help oversee every step. H2A, H2B, H3, and H4 are histone proteins. The histone chaperones assist in formation of tetrasome from the heterotetramer of H3-H4 on DNA. The tetrasome in combination with the H2A-H2B dimer forms the nucleosome. Histone chaperones are so important because without their help, positively charged histones would form aggregates with negatively charged DNA. The fact that the histones have a hydrophobic part and a slightly acidic part makes the protein even more attracted to DNA. Histone chaperones, although not sequentially similar, can be structurally grouped as beta-sheet sandwich chaperones, alpha-earmuff chaperones, beta-propeller chaperones, and beta-barrel and half barrel chaperones.
Beta-sheet sandwich chaperones
This structure is characterized by an N-terminal core domain of Saccharomyces cerevisiae Asf1 and the absence of an acidic tail. Histone binding was confirmed by mutagenesis and NMR chemical shift to occur at the hydrophobic and acidic surface of Asf1. The C-terminal tail of H4 can bind to either the yAsf1 beta-sheet or H2A mini beta-sheet. Therefore, yAsfi could access the H3-H4 dimer and form a complex that is structurally conserved. The crystal structure of human ASF1a reveals this complex. Asf1 passes off the H3-H4 dimer to other histone chaperones such as CAF-1 or HIRA. Asf1 regulates transcription, replication and repair. In addition to Asf1, Yaf9 is another chaperone in the beta-sheet sandwich chaperone. Yaf9 plays a role in H2AZ acetylation and deposition into euchromatic promoter regions. It also contains structurally conserved features that could serve as H3-H4 binding sites and regulates transcription.
Alpha-earmuff chaperones
This group is responsible for histone delivery from cytoplasm, binding to H1 and assembling and disassembling nucleosomes. These chaperones are characterized by their use of long alpha-helix for dimerization and linking alpha beta earmuff motifs. Mutagenesis confirms the histone binding site at the central and bottom surfaces of the earmuff domains. Vps75 is one of the chaperones in this group and it binds H3-H4 dimer. It is involved in the acetylation of H3 K56, which in turn aids the packing process. Along with acetylation, Vps75 is also responsible for regulating transcription, repair, and maintaining telomere length. The earmuff domains are closer in Vps75 than NAP1. NAP1 is responsible for transcription, H2AZ exchange, linker histone deposition, and histone delivery.
Beta-propeller chaperones
This group is characterized by acidic patches that are not as distinct as other chaperones. Nucleoplasmin is responsible for regulating nucleolar events and histone storage during such events as oogenesis, sperm chromatin decompaction, and nucleosomal assembly. Nucleoplasmin’s pentamer N-terminus has the ability to self associate into a decamer. The nucleoplasmin core works in conjunction with linker histones and core histones to produce five histone octamers. Along with having a beta-propeller chaperone structure, CAF-1 and RbAp46 has an alpha-helix at the N-terminus. These chaperones are negatively charged at the top and hydrophobic at the bottom. H4 alpha helix binds between the chaperones’ alpha helix and a binding loop in an acidic region. Consequently, the H3-H4 dimer structure is disrupted. To counteract, these chaperones take on the role of associating the H3-H4 dimer with several chromatin modifying complexes such as HAT1, PRC2, NURD, and NURF.
Beta-barrel and half barrel chaperones This family consists of FACT subunits Spt16, Pob3, Nhp6, SPT16 and SSRP1. FACT is the term used to basically mean that these chaperones facilitates chromatin transcription. Pob3’s structure consisting of a helix-capped beta-barrel helps it bind to the yeast replication protein A (RPA) complex to assist in replication. Spt16’s has linked aminopeptidase and pita-bread domains. Rtt106 binds histones acetylated on H3 K56. Its H3-H4 binding site is located in a loop in the C-terminal domain.
Histone variant chaperone Chz1
This histone chaperone group is characterized as not having a defined sequence or structure. Chz1 is a H2AZ-H2B binding protein. NMR confirms Chz1’s unique structure of alpha-helices that bind mainly on one surface of the H2AZ-H2B dimer with high affinity. Therefore, it dissociates more slowly than it associates.
The oligomeric state of the histone chaperone depends on the stage of the nucleosome assembly. Histone chaperone-histone binding could either be simple or multimeric. The weak individual binding adds up to a high affinity multimeric binding. Although Asf1 and Chz1 are both monomeric, they play different roles in nucleosome assembly. Asf1 prevents H3-H4 tetramer formation. H3 would usually dimerize inside the histone octamer, but Asf1 binds to H3 making it exposed to other histone chaperones. Once Asf1 is released, the H3-H4 tetramer can form with Rtt106 and CAF1 attached. It is after this stage that the tetramer is deposited onto DNA. Chz1 on the other hand binds to an already exposed histone octamer surface of H2AZ-H2B. Therefore, the histones are directly deposited on tetrasomes.
    Alpha beta earmuff chaperones assemble nucleosomes in vitro. NAP1 not only assembles histone octamers but also tetrasomes. NAP1 assembles histone octamers through H2A-H2B or
H2AZ-H2B. NAP1 assembles histone tetrasomes through H3-H4 and DNA. This process is not as favorable when there is H2A-H2B around. Vps75 is different from NAP1 in that it is more specific for H3-H4 and has weaker affinity for other histones. NAP1 binds all the histones that have the common histone fold through one surface. This is also the point in which it differs with FACT because FACT binds multiple surfaces of multiple histones. FACT carries out different functions depending on if it’s in vitro or in vivo. When it’s in vitro, FACT removes the H2A-H2B dimer. However, when it’s in vivo FACT puts the H2A-H2B dimers on DNA during the processes of transcriptional elongation, repairs, and replication.
    There’s only one binding site for H4 in dCAF-1 p55 and hCAF-1 RbAp48 subunits but there’s multiple binding sites for H3. The single binding site for H4 is on the opposite face of the H3-H4
dimer. This allows the CAF-1 subunits to either reach histone dimers bound to ASf1 or histone tetramers that are being deposited onto DNA.
    Np is assembled as a dimer of pentamers. Each face of these histone chaperones could bind an octamer. Np binds H2A-H2B dimers and N1 binds H3-H4 tetramers to form octamers. NASP,
nuclear autoantigenic sperm protein, binds H3/H4 and aids the nucleosomes assembly process.
Histone chaperone-guided folding pathways
Histone chaperones are extremely important in guiding the histone-DNA folding process because without it the histone would form intermediates that act as kinetic traps. Nucleosome assembly is an energetically favorable process and to remain that way the histone chaperones need to prevent these kinetic traps that prevent histones from ever binding on DNA. These kinetic traps are low in energy and it is not energetically favorable for histone to get out of these kinetic traps and bind on DNA when it is more stable in these intermediates. The histone chaperones allow histone-DNA complex to fold in a manner that is most stable and low energy. It is an energetically downhill process to get to DNA kinetic traps along the pathway are lower in energy so without the help of histone chaperones the histone would be stuck in that intermediate and never reach its destination. Histone chaperones work together with ATP-dependent chromatin remodelers. They are useful in times of kinetic traps to get the histones back on the correct pathway. Since kinetic traps are low in energy, chromatin remodelers get these intermediates out by raising its energy. These chromatin remodelers are also useful in breaking histone-DNA bonds. Certain acetylation marks on histones either increase or decrease affinity of the binding and is removed depending on if the nucleosomes needs to undergo assembly or disassembly.

## Nucleosome Remodeling

ATP-dependent chromatin remodeling complexes (remodelers) use the energy of ATP to modify the structure of chromatin.
There are four main families of remodelers:
* SWI-SNF
* ISWI
* CHD
* INO80/SWR
One SWI-SNF remodeler known as RSC can stimulate RNA polymerase II transcript elongation through a mononucleosome in a simple reconstituted chromatin transcription system. This is enhanced by histone acetylation as it increases the affinity of RSC for the nucleosome. The remodeler Chd1 is associated with chromatin at sites of active transcription, and it also plays a role in transcript elongation as it interacts with elongation factors Paf, DSIF, and FACT.

### Histone Modifications

Covalent modification of histones is another way of modifying chromatin structure and it that does not involve histone removal and replacement. Instead it involves altering the packaging of chromatin by affecting internucleosomal contacts or changing electrostatic charge. As well as using the covalently attached moieties as a binding surface for elongation associated effector complexes.
The aforementioned can be achieved using these three mechanisms:
* Histone acetylation
* Histone methylation
* Histone ubiquitation

# Reference

Selth, Luke A. Sigurdsson, Stefan. Svejstrup, Jesper Q. "Transcript Elongation by RNA Polymerase II". Annual Review of Biochemistry 2010. Vol. 79: 271-293. 04/01/2010. DOI: 10.1146/annurev.biochem.78.062807.091425
* 1 2 3 4 5 6 7 8 9 10 11 12 http://dx.doi.org/10.1016/j.tibs.2010.04.001