Monday, June 7, 2010

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.)  

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