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 TFIIB-Recognition Element (BRE), approximately 5 nucleotides upstream (BREu) and 5 nucleotides downstream (BREd) 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) 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.

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.

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