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.)  

Bacterial Transcription: Initiation, Polymerization, Termination

After the last post, I thought I would be updating more often, but that just didn't happen.  Either way, here's a post about bacterial transcription!

Bacteria as an Experimental System

Bacteria are a common genetic system, but why exactly do we use these tiny organisms to perform so many experiments?  Because they’re easy to use, of course.  There area  number of benefits to using bacteria, including:

- Establishing basic biological principles

- Genetic manipulation

- Short generation time

- Simple growth conditions

-  High population density 

- Ability to witness rare events

- Ability to select for rare variants

Bacterial Transcription

Transcription is function that has been heavily studied in bacteria.  This first step in gene expression is facilitated by a single RNA polymerase of six subunits.  Eukaryotes, in contrast, have four polymerases (I, II, and III, as well as a mitochondrial or chloroplast polymerase).  In bacteria and eukaryotes, the initiation of transcription requires a complex of proteins to assemble and facilitate polymerization of RNA from DNA templates.  

Bacterial RNA polymerase consists of six subunits, as mentioned previously.  The β and β’ subunits perform the polymerization reaction.  Two α subunits regulate the frequency of initiation.  The ω subunit is involved in stability and assembly of the polymerase enzyme. 

RNA polymerase first binds to the promoter region of DNA using a σ factor, which binds two specific regions of the promoter. The core polymerase and σ factor slide along DNA until they come upon a promoter.  This closed complex that finds the promoter converts to an open complex (not requiring any ATP for this action), which favors the separation of the DNA strands.  At this point, RNA polymerase begins to make short RNA segments, as it “stutters” along the DNA.  Small RNA oligos are formed, and σ factor begins to dissociate from the polymerase enzyme.  At this point, elongation of RNA transcripts can occur, which results from a tightening of the clamp and the formation of the RNA exit channel.  During elongation, RNA polymerase adds nucleotides to the growing RNA transcript at a rate of about 50 per second.  With σ factor dissociated, the “rudder” of RNA polymerase pries the DNA/RNA hybrid apart. 

When RNA polymerase is to stop the transcription of a gene, it has a few options.  First, the gene itself may have an AT-rich region that forms secondary structures that inhibit transcription after they have been copied.  These hairpin secondary structures may open the exit channel, and due to the less stable A-U base-pairing between the DNA and RNA, the transcript is released.  Additionally, there is a rho-dependent transcriptional termination method.  Rho is a hexameric protein that wraps approximately 60 bp of mRNA.  Rho, once bound to mRNA, activates and uses its ATPase activity to move as an RNA-DNA helicase.  Once it has become active, rho begins to unwind the RNA from the DNA, and when it approaches the active site of RNA polymerase, the transcript is released from the DNA. 

Bacterial genes in general can be found  in either direction on the genome and are very rarely overlapping.  RNA polymerase recognizes a distinct region on the chromosome to initiate transcription.  To identify this site, DNA footprinting is used.

DNA footprinting:

1.      Bind RNA polymerase to a DNA strand of known length

2.      Randomly cleave the DNA by nuclease or chemical agents

3.      Remove RNA polymerase from the DNA

4.      Separate the DNA strands on an agarose gel.

By DNA footprinting, it was recognized that there is a specific region that is “empty” (the footprint) on the agarose gel corresponding to where RNA polymerase binds.  Genetic analysis has identified two regions where RNA polymerase binds: at -35 and -10 relative to the initiation site.  The consensus sequences are TTGACA and TATAAT, respectively.