Part 3 of 3. Of part 1 of 4. So I guess it's like part 3 of 12, but that sounds too intimidating. Let's stick with 3 of 3.
RNA Polymerase II
RNA polymerases in general consist of about 10 subunits and making a protein of greater than 500 kDa. Five subunits are common to all of the three polymerases. However, RNAPII contains the all-important C-terminal domain (CTD): YSPTSPS, which is repeated 52 times in mammals (26 times in yeast). RNAPII that can initiate transcription has a CTD that is unphosphorylated, but upon initiation and movement of the polymerase from the promoter, the CTD becomes phosphorylated. RNAPII alone, however, is not enough to initiate transcription, as it requires a number of other factors for transcription actually begin. These include six GTPS: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Once these and RNAPII have assembled at the promoter, the pre-initiation complex (PIC) has formed, which allows for basal transcription. How often this PIC is formed is regulated by upstream activator and repressor proteins.
Motifs Required for Basal Transcription
A number of DNA sequences are necessary for the core promoter to actually lead to transcription of a gene:
The TATA box: located at about -25, it binds the TBP and is found mainly in tissue-specific genes. Consensus sequence of TATA(A/T)AA(G/A). This element is involved in positioning RNAPII to start transcription, so any mutations in this region can be devastating to transcriptional activity.
The BRE (TFIIB response element): located at about -32 to -35, binds TFIIB
The INR (initiator): located at -2, binds TFIID, and can stimulate TATA box activities, though weakly. Used by about 65% of genes in place of a TATA box.
The DPE (downstream promoter elements): located roughly from +28 to +32 and stimulate gene transcription.
The Steps in Transcription Initiation
Formation of the preinitiation complex (PIC) is the initial step in transcriptional initiation and involves the assembly of GTFs on the gene:
- TBP binds the minor groove of the TATA box, causing a bend in the DNA and promoting the binding of more factors
- About 10 TAFs bind TBP to form TFIID
- TFIIA binds TFIID complex
- TFIIB binds the TFIID-TFIIA complex
- TFIIF recruites RNAPII to the promoter
- TFIIE and TFIIH join to form the functional PIC
TAFs are a diverse set of proteins that affect the ability of TBP to interact with the promoter, and these TAFs are particularly important when there is no TATA box on the gene. These proteins can act as co-activators, functioning to recruit TFIID or interact with other transcription factors, for example. Additionally, other TAFs have acetyltransferase, kinase, and ubiquitin-conjugating activities.
Mediator is a large protein complex that stimulates or inhibits the activity of RNAPII. Other activators and inhibitors of transcription interact with mediator, sometimes at a long distance, and these signals are integrated to promote or inhibit RNAPII activity. While not all subunits of mediator are necessary for transcription, some are required.
After the formation of the PIC, transcription begins and the promoter is cleared, at which point the CTD on RNAPII is phosphorylated and the GTFs are released, except for TBP.