Transcription in Eukaryotes (Lectures 9-12)

RNA Polymerases

• Prokaryotes: There is one RNA polymerase

• Eukaryotes: 3 distinct RNA polymerases with specialized binding affinity for different classes of genes

Pol 1

• A distinct RNA polymerase in eukaryotes with a binding affinity for major ribosomal RNAs (rRNA: 5.8S, 18S, 28S)

• Makes large rRNA precursor in nucleolus

Pol II

• A distinct RNA polymerase in eukaryotes with a binding affinity for genes encoding proteins

• Makes heterogeneous nuclear RNA (hnRNA) and small nuclear RNA

Pol III

• A distinct RNA polymerase in eukaryotes with a binding affinity for genes encoding 5S rRNA, tRNAs (for translation), small nuclear RNAs for RNA splicing

Alpha-amanitin

• A toxic cyclic peptide found in mushrooms

• An inhibitor for Pol II and III so will no longer create essential RNAs, showing why certain mushrooms are toxic and deadly

• Image shows that even with very small amounts, activity for Pol II and III is greatly limited

Pol II Structure

• 12 subunits and has been sequences in yeast

• 3 subunits resemble the core subunits of bacterial RNA polymerases in both structure and function

• 5 are found in all three nuclear RNA polymerases, 2 are not required for activit and 2 fall into none of these categories

How were the RNA Polymerase subunits tagged?

• A epitope tag is genetically added to one subunit of the yeast RNA polymerase while all other subunits are normal, forming an active polymerase

• An antiepitope antibody is used against the tag, which immunoprecipitates the whole RNA polymerase

• Gel electrophoresis separates the denatured subunits

What are the different roles of Rpb1 subunit?

• Gene that encodes that largest subunit of Pol II

• Subunit IIa is the primary product in yeast but can be converted to:

  • IIb by the removal of CTD (7-peptide repeat)

  • IIo through phosphorylating 2 serine in the repeated heptad of the CTD

• IIa binds to the promoter

• IIo is involved in transcription elongation

Enhancers/Silencers

• Distal elements, far from start, ± 50 kb from start

• Position and orientation independent DNA elements that stimulate or depress

• Often tissue specific; rely on tissue specific DNA-binding proteins for their activities

• Some DNA elements can act either as a enhancer or silencer depending on what it is bound to

Promoter

• Proximal element, upstream of start, -200 to -40 bp

• Helps attract general transcription start site and direction of transcription

Core promoter

• Very close to the starting point, minimal region required fro accurate initiation of transcription

• Attracts general transcription factors and RNA polymerase II at a basal level and sets the transcription start site and direction of transcription

• Modular and can contain almost any combo of the following:

  • TATA box

  • TFIIB recognition element (BRE)

  • Initiator (Inr)

  • Downstream promoter element (DPE)

  • Downstream core element (DCE)

  • Motif ten element (MTE)

• TATA-less promoters tend to have DPEs

• Highly specialized genes tend to have TATA boxes

Class II promoters

• Recognized by RNA polymerase II

  • Core promoter

  • Proximal promoter

Enhancer

• Act through proteins that are bound to them; called activators

• Stimulate transcription by interacting with other proteins called general transcription factors at the promoter that promote the formation of a pre initiation complex

• Frequently found upstream of the promoter (not always though)

Silencers

• DNA elements that can inhibit (at a distance) transcription

• They work by causing the chromatin to coil up into a condensed, inaccessible and inactive form preventing the transcription of neighboring genes

Transcription in Eukaryotes

• RNA polymerases are incapable of binding themselves to their promoters so they rely on proteins called transcription actors to guide them

  • general transcription factors and gene-specific transcription factors (activators

General transcription factors

• combine with RNA polymerase to form a pre-initiation complex that is able to initiate transcription when nucleotides are available

• tight binding involves formation of an open promoter complex with DNA at the transcription start site that has melted

DABpolFEH (Class II Pre-initiation) Complex

• RNA polymerase II

• General transcription factors: TFIIA, TFIIB, TFIID, TFIIF, TFIIE, TFIIH

  • TF and polymerase bind in a specific order

Order of TF binding in DABpolFEH

• TFIID with TFIIA binds to the TATA box forming the DA complex

• TFIIB binds next, generating the DAB complex

• TFIIF helps RNA polymerase bind to a region -34 to +17 bp, forming the DABpolF complex

• The TFIIE and TFIIH bind to form the complete pre-initiation complex DABPolFEH

  • In vitro- participation of TFIIA seems to be optional

Structure and Function of TFIID

• TATA-box binding proteins (TBP)

• Highly evolutionarily conserved

• Binds to the minor groove of the TATA box

  • Saddle-shaped TBP lines up with DNA, underside of the saddle forces open the minor groove

  • TATA box is bent into 80° curve

  • TBP-associated factors (TAFs) specific for class II

What side does TBP bind to TATA box

• Inosine (I) and adenine look alike in the the minor grove, but different in major

• Thymidine and cytidine look identical from minor groove

Use of TBP

• Universal transcription factor required by Class I, II, III genes

  • TBP mutant cells do not transcribe these genes

• Require transcription of at least some genes of Archaea, single celled organisms lacking nuclei