RNA Polymerase and Basal Transcription

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:

1. TBP binds the minor groove of the TATA box, causing a bend in the DNA and promoting the binding of more factors
2. About 10 TAFs bind TBP to form TFIID
3. TFIIA binds TFIID complex
4. TFIIB binds the TFIID-TFIIA complex
5. TFIIF recruites RNAPII to the promoter
6. TFIIE and TFIIH join to form the functional PIC

TFIIH acts as a helicase to promote initiation and also has kinase activity to phosphorylate the CTD of RNAPII for promoter clearance.

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.  

More about Eukaryotic Transcription Initiation

Is it just me or does eucaryotic just look funny without the k?

Compared to prokaryotes (discussed previously), transcription in eukaryotes is complicated due to chromatin, multiple complexes, regulatory proteins, and a lack of transcription-translation coupling (one is in the nucleus; one is in the cytoplasm).  The complex that general transcribes genes into RNA is RNA polymerase II, which binds to specific sequences on the eukaryotic genome.  Genes in eukaryotes have several components, including enhancers, promoters and proximal elements, the TATA box, and the exons and introns of the gene.  The regulatory sequences surrounding a gene determine its transcription and utilization, accounting for temporal and spatial regulation of gene transcription. 

Specific factors are involved in the initiation of eukaryotic transcription.  Basal transcription factors (GTFs)  are required for transcription from all promoters, regardless of tissue-specificity.  RNA polymerase II (discussed above) is also required for transcription.  TATA-binding protein (TBP), which binds the TATA box, is also important in initiation of transcription, as are TBP-associated factors (TAFs) and coactivators of transcription. 

How do we analyze the activity of regulatory regions of genes?  Reporters, such as luciferase, GFP, or β-galactosidase are reporters, which can be used to measure the amount of transcription from a promoter or regulator element.  By placing a promoter or enhancer upstream of one of these reporters on a plasmid, transforming this plasmid into a cell, and measuring the amount of reporter gene transcribed, one can analyze the promoter activity.  The total amount of reporter protein that is synthesized is directly related to the activity of the promoter. 

Before being able to perform these reporter assays, however, the DNA sequences that regulate transcription must be putatively identified.  This can be accomplished via 5’ deletion analysis, in which DNA fragments upstream of the 5’ untranslated region (UTR) of a gene are introduced to a reporter vector.  As described above, the activity of the promoter is measured as a function of the reporter protein, such as luciferase. 

Additionally, one can perform linker scanning analysis, in which regions of the DNA are mutated with synthetic linker DNA.  These mutations should abolish the activity of the particular region of DNA that they “cover,” and the changes due to these mutations can be analyzed with reporter genes.  Now, with so many regions mapped and analyzed, bioinformatics can be used more frequently to identify shared enhancer sequences.

The core promoter of a gene consists of the site at which RNA polymerase II (RNAPII) binds and initiates transcription.  This site is approximately 35 bp upstream or downstream of the transcription initiation site, which allows RNAPII to interact with the basal transcription machinery. 

(Note: What about RNA polymerases I and III?  RNAPI is involved in the production of rRNA, and RNAPIII with tRNA.  RNAPII is highly abundant and is inhibited by α-amanatin, which interferes with the translocation of RNA and DNA and is found in poisonous mushrooms.  RNAPII synthesizes approximately 50% of the RNA in an active cell.)