regulation of gene expression in eukaryotes: transcription

prokaryotic vs eukaryotes gene expression regulation levels

prokaryotes

1. transcription → primary

2. RNA processing

3. translational

4. post-translational

eukaryotes

1. chromatin structure and epigenetics

2. transcription → primary

3. RNA processing

4. RNA export

5. RNA stability

6. translation

7. post-translational protein modifications

8. protein stability

prokaryotic vs eukaryotes gene expression regulation

• primary control of gene expression is on the transcriptional level for both prokaryotes and eukaryotes

• transcription and translation occurs simultaneously in prokaryotes

• separation of transcription and translation in eukaryotes allows for additional controls

• greater complexity and organisation in eukaryotes

one RNA polymerase transcribes all (?) genes

prokaryotic

(?) factor positions the polymerases at the promoter

sigma

prokaryotic genes are often (?)

polycistronic → encode for multiple proteins

polycistronic genes are expressed under the control of (?) promoter

one

prokaryotic genes often involve simple regulatory sequences consisting of a (a) and a (b)

(a) promoter

(b) operator

eukaryotic genes are usually (?)

monocistronic → encode for one protein

prokaryotic vs eukaryotic genes

• polycistronic vs monocistronic

• eukaryotic genes have introns

• complex vs simple promoters reflecting complexity of organism

• coordinated expression of a set of proteins that are required at the same time requires coordinated regulation of those genes

eukaryotes use three different RNA polymerases (name and describe)

1. RNA polymerase I

• transcribes rRNA genes: 5.8S rRNA, 18S rRNA AND 28s rRNA genes

2. RNA polymerase II

a) transcribes mRNA genes

b) aids mRNA splicing

c) guides chemical modifications of RNAs (e.g. tRNAs)

3. RNA polymerase III

• transcribes tRNA genes: 5S rRNA gene, some snRNA genes

each eukaryotic RNA (?) requires different promoter elements and different transcription factors

polymerase

a composite core promoter containing a TATA box and initiator sequences just upstream of the transcription start site → what binds here?

general transcription factors and RNA polymerase II

proximal promoter elements within 200 base pairs of composite core promoter → what binds here?

cell or tissue specific transcription factors

downstream promoter elements (DPEs) → what binds here?

some general transcription factors

distal promoter elements → what binds here

• enhancers → activating transcription factors bind here

• silencers → gene repressing transcription factors bind here

insulator action

prevent distal enhancers from acting on the promoter of neighboring genes or prevent spread of heterochromatin

eve gene expression

• eve gene expression is controlled by several regulatory DNA segments → each binding different regulatory proteins

• the regulatory segment that directions the formation of stripe two in the embryo (B) can be excised from the DNA and inserted upstream of the E. coli lacZ gene → encodes for B-galactosidase

• B-galactosidase can convert a colourless substrate into a blue product → ‘reports’ the regulation of gene expression mediated by segment 2

• when the reporter fusion DNA is introduced into the genome of a fly (D), the blue colour shows where the resulting embryo expresses B-galactosidase → precisely in the position of the second eve stripe

general TFs position RNA polymerase (?) close to the transcription site, this is similar to the role of the prokaryotic sigma factor

II

the assembly of general TFs and the eventual RNA polymerase II occurs at what site along the DNA in eukaryotes?

core promoter

the TATA binding protein (TBP), a subunit of TFIID, binds to the (?) in the core promoter and bends the DNA

TATA box

explain what is occurring in this diagram

1. general TFs position RNA polymerase II close to the transcription site

2. the assembly of general TFs occurs at the core promoter of protein encoding genes

3. the TATA binding protein (TBP), a subunit of TFIID, binds to the TATA box in the core promoter → bends the DNA

4. TFIID also binds the transcription start site and downstream promoter elements

5. TFIIB, TFIIE and TFIIH sequentially assemble at the core promoter

6. TFIIF positions the RNA polymerase II at the transcription start site → preinitiation complex

7. TFIIH binds and uses energy to pull apart the DNA double helix → template strand exposed

8. TFIIH phosphorylates RNA polymerase II → allows transcription to begin

9. most of the general TFs dissociate from the core promoter

cell or tissue specific transcription factors

• TF DNA binding domain → amino acid sequence that interacts with DNA

• cell/tissue specific TFs bind via non-covalent interactions between amino acid side chains and DNA → major groove of DNA

• bind to proximal, distal, and downstream promoter elements

• often bind as dimers → two TF proteins

• the TF dimer also contains an activation or repression domain → amino acid sequence that regulates gene expresion

the regulatory effects of many TFs is mediated by the mediator complex

• a multiprotein complex that brings the activities of transcriptional regulator together so that they can aid transcription by helping to assemble:

1. RNA polymerase II

2. general transcription factors

3. chromatin remodeling complexes

4. histone modifying enzymes

a typical eukaryotic gene is controlled by (?) TFs

many

combinational control of eukaryotic gene expression

• many transcription factors are active at any one time in a cell and can interact with:

• RNA polymerase

• each other → homodimer

• other TFs → heterodimer

• TFs can act as activators, co-activators or repressors of transcription

• repressors of transcription interfere with the binding of a TF to DNA elements

• the combination of TFs bound to regulatory DNA elements determines the outcome of gene expression

transcription factors can be regulated (explain using cortisol as an example)

1. gene 1,2 and 3 have TFs bound to their respective promoter elements

2. these TFs are not sufficient to activate transcription effectively → low gene expression

3. the presence of cortisol activates the cortisol receptor forming a cortisol receptor complex

4. activated cortisol receptor complex binds to the cortisol receptor binding sequence

5. this sequence is found in promoters of the three genes

6. this activates efficient transcription of the three genes at high levels

histone modifying proteins can change (?) structure

chromatin → by influencing the accessibility of DNA to transcription factors e.g. exposure of the TATA box

chromatin remodeling complexes can change chromatin structure by:

1. activating transcription regulators → recruit chromatin modifying proteins via the mediator complex →open up chromatin → transcription

2. repressing transcription regulators → recruit chromatin modifying proteins via the mediator complex → close chromatin → no transcription

DNA methylation

addition of methyl groups to DNA via DNA methyltransferases

in vertebrates, DNA methylation is confined to selected cytosine nucleotides next to a (?)

guanine

methylation is symmetrical

methyl group added to both DNA strands

methylation of cytosines in gene promoters prevents:

binding of transcription factors

methylation of cytosine recruits (?) modifying enzymes

chromatin → e.g. histone deacetylases which causes condensed chromatin

less methylation results in (?) condensed chromatin

less → therefore gene expression turned on → transcription

de novo methylation

regulate gene expression by methylating non-methylated cytosines on both DNA strands

histone modifications and DNA methylation are examples of:

epigenetic modifications → heritable, reversible and do not change the DNA sequence

epigenetic differences in identical twins

• begin life with the same genetic information including epigenetic tags

• over a lifetime the twins’ environment will diverge

• resulting in individual epigenetic tags forming in each twin

• difference in the epigenome is what makes them become different as they age

environment and diets produce (?) changes that affect growth, development and long-term health

epigenetic

DNA methylation in normal vs cancer cells

• normal cells → CpG islands residing in promoter regions of genes are often hypomethylated/unmethylated → genes expressed

• cancer cells → CpG islands become hypermethylated

• which results in silencing of their associated genes → e.g. tumor suppressor genes are often silenced in cancer cells due