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

Cis-acting elements

• Regulatory DNA sequences on themselves, regulation by the Sam molecule that is being regulated

Trans-acting elements

• Regulation is by a different molecule that is being regulated

• enhancers: activators bind and its affects transcription, often far way, in another gene

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

TAF1, TAF2

• TBP-Associated Factors

• Help the TFIID bind to the initator of the DPE of promoters

• Enable TBP to bind to TATA-less promoters that contain elements such as GC box

Enzymatic activities of TAF1

• Histone acetyletransferase (HAT)

• Protein kinase

Structure and Function of TFIIB

• Binds to TBP at the TATA box via C-terminal domain and polymerase II via its N-terminal domain

• Important role in establishing transcription start site

TFIIH

• Last TF to going pre-initiation complex

• Structure

  • 4 subunits: protein kinase

  • 5 subunits: Core with 2 DNA helicase

• Roles

  • Phosphorylates CTD of RNA polymerase II

  • Unwinds DNA at the transcription start site to create transcription bubble

  • Expansion releases the stalled RNAPII and allows it to clear the promoter

How is IIO (phosphorylated form of polymerase enzyme) created?

• Pre-initiation complex forms with hypophosphorylated form of RNA polymerase II (IIA)

• TFIIH phosphorylates serine 5 in CTD (carboxyl-terminus domain (Tyr-Ser-Pro-Thr-Ser-Pro-Ser)) of the largest subunit creating the phosphorylated form

• Essential for initiation of transcription

How does serine phosphorylation change when there is a shift from initiation to elongation?

• transcription: TFIIH phosphorylates serine 5

• elongation: CTDK-1 phosphorylates serine 2

Activators

• Gene-specific transcription factors that bind to enhancers to provide an extra needed boost to transcription

• Also recruit RNA polymerase to promoters

• Stimulate binding of general transcription factor and RNA polymerase to a promoter

TF Protein structure

• DNA binding domain

• Activation/repression domain

• Often dimerization domain

• Sometimes binding sites for hormones that regulate the TF

Zinc Fingers

• Type of TF

• An antiparallel beta-sheet and an alpha-helix

• Two cysteines in the beta sheet and two histidines in the alpha-helix coordinate the zinc ion in the middle

• Zinc does not bind to DNA

  

Gal4 DNA binding

• yeast activator

• Has a zinc finger in the first 60 aa to coordinate DNA binding

• Contains a DNA-binding motif with six cysteines that complex two zinc ions

• each monomer contains dimerization (two molecules combine to form a larger molecule called a dimer) domain

bZIP (leucine zipper) transcription factor and bHLH

• One part of the domain contains a region that mediates sequence specific DNA binding properties and the leucine zipper that is required to hold together (dimerize) two DNA binding regions.

• Binding sites in major grove of DNA

• Must have another domain to bind RNA polymerase

• Leucine on hydrophobic faces of helices interact to enable dimerization

Hypotheses for RNA polymerase recruitment to promoter

1. General TF causes a stepwise build-up of pre-initiation complex

2. General TF and other proteins are already bound to polymerase in a complex called RNA polymerase holoenzyme

Hypothesis #2 model using GAL11P-containing holoenzyme

• Dimerization domain of GAL4 binds to GALL11P in holoenzyme

• After dimerization, holoenzyme and TFIID binding to the promoter

• this would not work with GALL11 as it does not bind to GAL4 like GAL11P does

• instead LexA DNA binding to GAL11 mimics transcriptional activation activity of GAL11P

Whole process of gene activation by RNA Polymerase II

• RNA Polymerase II is recruited to form the pre-initation complex

• Thousands of base pairs upstream, an activator bound to an enhancer loops to activate the RNA polymerase

• CTD is phosphorylating, initiating transcription

Chromatin Structure

Composed of DNA and proteins, histones

• Roughly equal masses of DNA and histones

• 1 histone octamer (8 histone proteins (2 of H2A, H2B, H3, H4into a disk)) per 200 bp of DNA

• Beads on a string form a chromatin

Chromatin Packing

Many levels of chromatin packing postulated to give rise to the highly conensed mitotic chromosome

Core histone structure

Histones 2A and 2B form a dimer, H3 and 4 form a dimer

Bending of DNA in a nucleosome (the histone and DNA)

• DNA makes 1.7 tight turns around the histone

• Minor groove is compressed on the inside of the turn

• 142 hydrogen bonds are formed between DNA and the histone core in each nucleosome

Role of histone H1

Binds to each nucleosome, contacting both DNA and protein, changing the path of the DNA as it exits from the nucleosome to help compact nucleosomal DNA

Histone impact on transcription

• Modifications to histones can impact transcription by making DNA more or less accessible to proteins

• DNA can be too tightly wrapped for proteins to access

Nucleosome sliding

• Allows proteins to find targets faster

• Catalyzed by ATP-dependent chromatin remodeling complexes (ISW1 or SWI)

• Uses energy from ATP hydrolysis to push on DNA and loosen attachment to nucleosome core

• read-writer complex can spread chromatin change along a chromosome

Histone methylation

• Covalent modification of core histone tails

• Adds 1,2, or 3 methyl groups to nucleosomal histones to either activate or repress transcription

• Typically modifies Lysine

Histone phosphorylation

• Covalent modification of core histone tails

• Adds a phosphate group

Histone acetylation

• Covalent modification of core histone tails

• Acetyl groups are added to lysine residues within histone tails, a key epigenetic modification that loosens chromatin structure and promotes gene expression

Histone Code

Hypothesis proposing that specific patterns of chemical modifications (post-translational modifications or PTMs) on histone proteins

Histone writers

Proteins that add histone markers

• Histone acetlytransferase

Histone erasers

Proteins that remove histone marks

• Histone deacetylase

Histone reader proteins

• Recognizes Histone modification marks

• Binds when a certain mark is present

• Can be involved in chromatin remodeling

  • Nucleosome displacement/relocation

  • Histone replacements

Model for histone code at human IFN-B promoter

• IFN-β is an inflammatory cytokine

  1. Activators recruit GCN5 (histone acetyltransferase) to acetylate H4K8 and H3K9

  2. Activators recruit a kinase to phosphorylate H3S10

  3. H4K8 attracts SWI/SNF (nucleosome slider) to remodel nucleosome

  4. Now allows the binding of TFIID which is now attracted by the TATA box but also H3K9ac and H3K14