Eukaryotic Transcription

Transcription in Eukaryotes

1. Eukaryotic DNA is packaged into chromatin, which in the condensed state (default state) results in a promoter that is inaccessible to RNA polymerase. Transcription can only occur when DNA is decondensed and dissociated from the histones around which it is wound.
2. Transcription and translation cannot occur at the same time because eukaryotes have a separate compartment for the DNA (nucleus).
3. Prokaryotic cells: genes with common function are arranged linearly and are transcribed together on a single mRNA (polycistronic).
4. Eukaryotic cells: genes with common functions can be scattered throughout the genome, on different chromosomes and can therefore be transcribed and regulated separately from one another.
5. Eukaryotic mRNA is always monocistronic: one mRNA = one protein.
6. Eukaryotes have three types of RNA polymerase:

• RNAP I: transcribes rRNA
• RNAP II: transcribes mRNA
• RNAP III: transcribes tRNA

1. Messenger RNA requires extensive modification before being translated into protein (maturing of RNA).

1. Promoters are more complex but also have a consensus sequence similar to that in prokaryotes. This is called the TATA box and is located about -30 nucleotides upstream of the transcriptional start site.
2. RNA polymerase needs the help of transcription factors to bind to DNA and start transcribing. Instead of a simple sigma factor, several transcription factors (TFs) bind to the promoter to recruit RNA Polymerase II. See below for more details on this.

Transcriptional Control

• The equivalent of sigma proteins in prokaryotic transcription are basal transcription factors in eukaryotic cells.
• Basal transcription factors include a TATA-binding protein (TBP) that binds to the TATA box in the promoter of eukaryotic genes. Following TBP binding, the RNA polymerase is recruited.
• Other transcription factors (such as enhancers) bind to different areas within the gene and interact with the polymerase to initiate transcription.
• Enhancers can even be located far away from the genes they affect.
• Transcription factors help to coordinate gene expression. Genes located on different chromosomes can be controlled simultaneously through the use of the same transcription factor.

General Transcription Factors Required for Initiation

• The CTD is made up of Tyr-Ser-Pro repeats (52 repeats in humans)
• During initiation of transcription, the CTD is unphosphorylated
• As the polymerase begins the elongation phase of transcription, the CTD is phosphorylated
• RNA processing proteins, like capping, elongation, and splicing factors can bind to the CTD depending on it's phosphorylation state

• TFIID consists of a TATA box Binding Protein (TBP) and 13 other TBP-Associated Factors (TAFs)
• TFIID forms a complex with TFII A and TFIIB and the TATA-box
• TFIIF and RNAP II then bind
• TFIIE binds and creates a site for TFIIH to bind
• TFIIH has helicase activity which unwinds the DNA
• After transcription has been initiated, TFIIH has kinase activity which phosphorylates the CTD of RNAPII

Regulation of Eukaryotic Transcription

• Activate transcription by binding to control regions on the DNA (enhancer sites, promoters, initiator sites, etc.)
• Transcriptional activators have a DNA binding domain and an activation domain
• DNA binding domain: Zinc finger domains (C2H2 or C4), leucine zipper, basic Helix-Loop-Helix
• Activation domains: short structures containing high percentage of one or two amino acids (e.g. Asp, Glu, Gln, Pro, Ser or Thr). Facilitates protein-protein interactions: may have a random structure until interaction with a co-activator protein

• Repress transcription by binding to control regions on the DNA
• Transcription repressors have a DNA binding domain and a repression domain
• DNA binding domain: Zinc finger domains (C2H2 or C4), leucine zipper, basic Helix-Loop-Helix
• Repression domain: short structures (<15 amino acids). Facilitates protein-protein interactions: may need to bind to co-repressor proteins to silence transcription

How does a finite set of transcription factors control the expression of so many genes??

• Heterodimers: combinations of different transcriptions binding together to interact with different DNA sequences.

• Inhibitory factors: Can block the activity of some transcription factor DNA binding domains
• Cooperative Binding: Unrelated transcription factors binding to neighboring sites
• Example: A transcription factor with weak repression activity binds nearby another weak repressor= STRONG repression

Heterochromatin vs. Euchromatin

Post-translational Histone Modification

• mono-, di- or tri- methylation on Lysine (K) or Arginine (R)
• acetylation on lysine (K)
• Phosphorylation on Serine (S) or Threonine (T)

• H3K4me3 causes chromatin to decondense= euchromatin
• H3K9me2 causes chromatin to condense= heterochromatin
• Acetylation of lysine usually correlates with euchromatin where as deacetylation correlates with heterochromatin