Post/Transcriptional Regulation in Eukaryotes

Transcriptional Regulation in Eukaryotes

Transcriptional regulation drives…

interspecies difference and many biological processes beyond

ex.) humans and chimps contain 96% of the same genes, however expression causes different phenotypes

Gene expression can be controlled at multiple levels

Alteration of Structure (methylation and acetylation of DNA and histones)

Transcription

mRNA processing (splicing, caps, and tails)

mRNA stability

Translation (inactive proteins)

Posttranslational modification (modified protein active and stable)

Eukaryotic vs prokaryotic gene regulation

• Eukaryotes are more complex than prokaryotes (nucleus, splicing, timing of translation, etc.)

• Regulation is also more complex than prokaryotes

  • Expression patter: when and where to express

  • Transcription initation is still a critical step

  • Control of RNA stability and processing are important

  • Regulatory state can be passed to daughter cells 

Key Regulatory Differeces Between Eukaryotes and Prokaryotes

Control of transcription through specific DNA binding proteins (pro-yes; euk-yes)

Reutilization of same DNA-binding motifs by different DNA-binding proteins (pro-yes; euk-yes)

• Activator proteins (pro-yes; euk-yes)

• Repressor proteins (pro-yes; euk-yes)

Specificity of binding to DNA by regulatory protein (pro-specific; euk-highly specific)

• Affinity of binding (pro-strong; euk-very strong)

• Role played by chromatin structure (pro-no; euk-yes)

Coordinate control achieved with operons (pro-yes; euk-rare)

• Differential splicing (pro-no; euk-yes)

• Attenuation (pro-yes; euk-no)

• mRNA processing (pro-no; euk-yes)

• Differential polyadenylation (pro-no; euk-yes)

Differential transport of RNA from nucleus to cytoplasm (pro-no; euk-yes)

RNA interference carried out by micro-RNAs (pro-no; euk-yes)

Eukaryote Pol II transcription initiation

1. TFIID transcription factor binds to the TATA box through TBP

2. Then transcription factors and RNA polymerase II bind to the core promoter

3. Transcriptional activator proteins bind to sequences in enhancers

4. DNA loops out, allowing the proteins bound to the enhance to interact with the basal transcription apparatus

5. Transcriptional activator proteins bind to sequences in the regulatory promoter and interact with the basal transcription apparatus through th mediator

Pre-initiation complex

RNA polymerase II and general transcription factors bind at core promoter

Eukaryotic gene regulation at transcription initiation (basal level)

Controlled by a complex of RNA polymerase, general transcription factors to carry out transcription (binds to Core Promoter)

Eukaryotic gene regulation at transcription initiation (normal)

other transcriptional factors (binds to regulatory promoter and enhancer)

Transcriptional Factor (TF), trans-acting proteins

• Bind DNA at a specific sequence (consensus) through on or more DNA-binding motifs (like the helix-turn-helix, zine finger (ZnF or ZNF), or leucine zipper)

• Second function is to interact with basal transcription apparatus to influence transcription rate

• Can be either activator or repressor

• Can recuit co-activators or co-repressors

• May have acetyltransferase activity and so stimulate transcription by altering chromatin structure

• TF binding to DNA is NOT permanent: dynamic

Regulatory Promoters and Enhances

• Promoter is at the near upstream position of TSS

• Enhancer is very distant (10s-100s of kb) from TSS

• Enhancer can lie either up or down stream of a gene

• Each gene has one promoter but many enhancers

• Both are a few hundreds base pairs long

• Both are cis-acting

• Both contain binding sites for several different TFs

• Both can be simultaneously bound by a mixture of activators or repressors

• Both sequence orientation can be reversed (either on top or bottom DNA strand)

Activator Proteins

Transcription factors that bind to enhancers and regulatory promoters (activator domain that interacts with other transcriptional regulatory proteins)

Responsible for much of the high levels of transcription of different genes

Increase transcription rates by interacting directly or indirectly with basal factors at the promoter

Mechanisms of activator effects on transcription

• Stimulate recruitment of basal factors and RNA pol II to promoters

• Stimulate activity of basal factors already assembled on promoters

• Facilitate changes in chromatin structure

Repressors can bind directly to

promoters and enhancers (most eukaryotic repressors do NOT directly block RNA polymerase)

Repressor Mechanism: Competition

Competition for binding between repressor and activator proteins

Binding of represssor to enhancer blocks binding of activator

Quenchinng Type I

Repressor binds to and blocks the DNA binding region of an activator

DNA-binding domain is blocked. Activator cannot bind to enhancer

Quenching Type II

Repressor binds to and blocks the activation domain of an activator

Activator can bind to enhancer, but cannot carry out activation

GAL system of Yeast

Model of Transcriptional Regulation in eukaryotes: encode enzymes needed to metabolize galactose

GAL genes are NOT expressed unless galactose is present

• Galactose induces 1000-fold expression of GAL1, GAL2, GAL7, and GAL10

• Each of the GAL genes has its own promoter

• Transcription of each structural gene is controlled by an enhancer/upstream activator sequence (UAS_G)

Example of Transcriptional Activator and Repressor: regulation of galactose metabolism in yeast

No galactose present: GAL80 blocks GAL4 from activating transcription

Galactose present: GAL3 inhibits the binding of GAL80 to GAL4 → activation of transcription

GAL4

Transcriptional activator that binds to UAS_G DNA

• Has activation domain and a DNA-binding domain

• When bound to UAS_G, it enhances the assembly of the basal transcription apparatus, maximize GAL genes’ transcription. GAL80 is a repressor that binds to the activation domain of GAL4

• When GAL80 binds to GAL4, GAL4 is not able to activate transcription

GAL3 Binds to GAL80 and inhibits its interaction with GAL4

Absence of galactose: GAL80 binds to the activation domain of GAL4 and blocks the activation of transcription

Presence of galactose: GAL3 binds to GAL80 and prevents GAL80 from binding to GAL4 → GAL4 stimulates the basal transcription apparatus

• Regulated by galactose

Insulators

Enhancers act on long range 

How to prevent regulation of non-target genes?

Insulators and Regulatory Neighborhoods

Insulators are specific DNA sequences that block the effects of enhancers in a position dependent manner

Located between enhancers

Specific proteins bind to insulator sequences (like CTCF binds to DNA sequences and prevents influence of activation/repression of adjacent genes)

Acts through 3D changes in DNA (looping)

May also inhibit the spread of chromatin structure alterations

AKA boundary elements

Topologically associating domain

large regions of spatially interacting chromatin, important aspect of transcriptional regulation

TADs are large loops of DNA with two proteins (CTCF and cohesin) at the base of the loops

Some: CTCF binds to insulators

Multiple genes can be within a TAD and have different interactions with an enhancer

Allow enhancers to interact with promoters within the loop but NOT with promoters outside of the loop

Coordinated gene regulation in eukaryotes

Multiple related genes respond to same stimulus because they share short regulatory sequences in their promoters or enhancers

Response element (aka, consensus sequence): DNA sequence that was recognized and bound by TF in response to stimulus

Single gene can be activated by different response elements: allow the same gene to be activated by different stimuli (physiological processes)

Complex Regulations Enable Fine-Tuning of Gene Transcription

• Each gene can be regulated by many regulatory proteins bound to multiple enhancers, which together controls the expression level and cell states: cell-type, development, proliferation, response to stimuli, etc.

• In humans ~2000 genes encode transcriptional regulatory proteins (tightly regulated and each protein can act on many genes)

• Enhancer can be bout by both activators and repressors with varying affinities

• Different sets of cofactors and corepressors compete for binding to activators and repressors

Posttranscriptional Regulation

RNA stability

• Increased by additions of 5’ cap and 3’ poly(A) tail

• RNA interference: small RNAs (20-30 nt)

Alternative mRNA splicing

Translational control

• Rold of small RNAs

Post-translational modification

• Ubiquitination

• Phosphorylation

Eukaryotic Gene Structure and pre-mRNA processing 

pre-mRNA

• Undergoes capping, splicing, and polyadenylation

Mature mRNA

RNA Splicing

• Removes introns

• Splicing apparatus recognizes sequences at splice sites

• Strongly regulated by sequence inside and outside of the introns

RNA splicing removes introns from precursor mRNA

During transcription precursor mRNAs are spliced to produce mature mRNAs

Exons

Gene sequences that are present in mature mRNA after removal of introns

• Can be very small (50 nt or even smaller)

Introns

Gene sequence that are present in pre-mRNA, but not in mature mRNA

• Can be huge (up to several hundred kb)

• Some eukaryotic genes have many introns

• Ex. dystrophin gene of humans (exons make up <1% of the gene)

RNA splicing

removal of intron from pre-mRNA

95% of all human genes with multiple exons are alternatively spliced

Happens in nucleus (spliceosome)

Splicing requires three sequences in the intron

Alternative splicing not always produce different proteins: depsn on where coding frame starts and ends

RNA splicing process

1. mRNA is cut at the 5’ splice site

2. 5’ end of the intron attaches to the branch point

3. A cut is made at the 3’ splice site

4. Intron is released as a lariat (transesterification)

5. Two exons are spliced together

6. Bond holding the larat is broken and the linear intron is degraded

7. Spliced mRNA is exported to the cytoplasm

Requires spliceosome containing small nuclear ribonucleoproteins (snRNP)

2’-5’ phosphodiester bond

Made between the G at the 5’ end of the intron and the A of the branch point and produces a lariat structure

Start of Intron

GU (mRNA)

GT (DNA)

Different Types of Alternative Splicing

Exon skipping

Alternative 3’ splice site selection

Alternative 5’ splice site selection

Mutually exclusive exons

Alternative mRNA splicing: SV40 gene produces two oncoproteins

1. Use of the first 5’ splice site produces an mRNA that encodes the large T antigen

2. Use the second 5’ splice site produces an mRNA that encodes the small t antigen

3. The SF2 protein enhances the use of the second splite site

Alternative mRNA splicing determines Drosophila sex phenotypes (XX)

1. XX embryos, the activated Sxl (sex lethal) gene produces a protein

2. causes tra pre-mRNA to be spliced at a downstream 3’ site

3. produces Tra (transformer) protein

4. Together, Tra and Tra-2 proteins direct the female-specific splicing of dsx (doublesex) pre-mRNA

5. Produces a protein that causes the embryo to develop into a female

Alternative mRNA splicing determines Drosophila sex phenotypes (XY)

1. In XY embryos, the Sxl gene is not activated, and the Sxl protein isn’t produces

2. Thus, tra pre-mRNA is spliced at an upstream site

3. Producing a nonfunctional Tra protein

4. Without Tra, the male-specific splicing of dsx pre-mRNA

5. Produces a male Dsx protein which causes the embryo to devlop into a male