Eukaryotic Gene Regulation – Module 5
WHAT IS GENE REGULATION
• Gene regulation = process of controlling which genes in a cell are
expressed/used to make a functional protein
• Expression in correct spaces = spatial regulation
• Expression at right time = temporal regulation
• Expression at right levels
• Function à required for the maintenance of homeostasis + allow response to
environmental, physiological and developmental signals (eg. Infection)
o Allow differentiation during embryonic development + positioning of
tissues/organs
• Cannot have all cells making all proteins because different cell types have different
functions and need different proteins
• Genes can respond to multiple signals and integrate response
• Regulation occurs at multiple levels in eukaryotes
Eukaryotes Prokaryotes
DNA packaged into chromatin that makes Naked DNA
it unavailable to transcription - Rely on repressors and activators
Additional RNA processing events Mature mRNA produced immediately
Transcription in nucleus and translation in Transcription and translation occurring
cytoplasm simultaneously in the cytoplasm
Regulation of transcription by the interaction of trans regulatory elements and cis
regulatory elements is the same for both eukaryotes and prokaryotes
REGULATION AT MANY LEVELS
1. Chromatin structure
2. Transcription
3. RNA processing
4. mRNA stability
5. Translation
6. Post translational modifications
1. CHROMATIN STRUCTURE
o DNA (146 base pairs) is complexed with histone octamer (2 copies of each
H2A, H2B, H3 AND H4) into nucleosomes
o Nucleosomes condense into chromatin
o Structure is dynamic and can be loosely packed or tightly
§ Controls accessibility of DNA for transcription
• Condensed chromatin – repressive
• Open chromatin = permissive
o Post translational modifications à recognized by chromatin remodelers to
uncondense and condense chromatin structure
§ Methylation (condenses)
§ Acetylation (decompresses)
• Eukaryotic DNA has two regulatory regions for genes
o Enhancers
§ Binding of regulatory transcription factors
, o Promotors – core and proximal
§ RNA pol binding to core promotor after general TF bind
2. TRANSCRIPTION
o 3 types of RNA pol in Eukaryotes
§ RNA pol 1
• Transcribes RNA components of ribosome
§ RNA pol 2
• Transcription of mRNA, microRNA, siRNA, snRNA
§ RNA pol 3
• Transcribes 5S rRNA and tRNA
• Primary RNA transcript produced is complementary to DNA antisense strand so that
the RNA transcript is a copy of the sense strand except that it has uracil not thymine
o Has exons and introns
o Poly A tail at 3’ end
• How does RNA pol find transcriptional start site?
o RNA pol on its own cannot recognize the start site
o Recruited by general transcription factors
§ GTF bind to core promotor (+- 30 nucleotides of transcription start site
– TSS)
o GTF recognize specific DNA motifs + histone modification patterns
o GTF bind to core promotor and recruit RNA pol to form the transcription
initiation complex (TIC)
o Transcription can then occur
• How was this discovered??
o EMSA à electrophoretic mobility shift assay
§ Tests for protein nucleic acid interaction
§ Uses concept of gel electrophoresis whereby molecules are separated
on size
• Apply current so as to cause nucleic acid to move through the
gel and be attracted to the positive electrode given its negative
charge due to the phosphate backbone
§ Add radioactive label to nucleic acid to allow for visualization in the
gel
• Label the nucleic acid not the protein
§ Uses small stretches of DNA (negative charge)
§ Mix DNA with proteins
• If no interaction à will see free nucleic acid at the bottom of the
gel (because small)
• If interaction à see a higher molecular weight band
o Protein slows movement of nucleic acid through gel
o Thickness of band corresponds to molecular weight of
protein
, • lane 6 – all 5 transcription
factors are present
o See a high number of
higher MW bands
o Adding one factor allows
you to see what factor binds
first (lanes 1-5)
§ TF2D binds first!!
o Causes a kink in the DNA
and then TF2B binds
§ Lanes 2-5 only show free
nucleic acid because they
cannot bind first
o Lane 7, all other factors
except TF2D added and only
the free nucleic acid is
present
§ Therefore, TF2D is
essential!
Result/Conclusion à no
direct binding of RNA pol
without transcription factors!!
• TF2D and TF2B recognize specific short sequence motifs in the core promotor (core
promotor = basal promotor)
§ Do not require all but at least some of these motifs are required in
euakryotes
§ Function = recruitment of GTF to allow RNA pol to bind and for
transcription to occur
§ Mutations would be lethal!!
o Eg. TATA/Pribnow box recognized by TF2D subunits
o Eg. TF2B recognition element
o Eg. Initiator
o Eg. Downstream promotor element
• Binding of GTF leads to kinks in DNA (bending) that are essential for transcription
o Caused by TF2D (contains TBP – Tata box binding protein)
• Transcriptional regulatory regions
o Enhancer – bound by regulatory transcription factors
o Proximal promotor – bound by regulatory transcription factors
o Core promotor – bound by general transcription factors (required for
transcription to occur)
*regulatory transcription factors determine whether or not transcription DOES occur
*different nuclei have different complements of regulatory TFs
*complement of regulatory TFs vary over time and space – allows for dynamic regulation!!
• Different genes have different binding sites
• Mis-expression of regulatory TFs can have drastic effects
WHAT IS GENE REGULATION
• Gene regulation = process of controlling which genes in a cell are
expressed/used to make a functional protein
• Expression in correct spaces = spatial regulation
• Expression at right time = temporal regulation
• Expression at right levels
• Function à required for the maintenance of homeostasis + allow response to
environmental, physiological and developmental signals (eg. Infection)
o Allow differentiation during embryonic development + positioning of
tissues/organs
• Cannot have all cells making all proteins because different cell types have different
functions and need different proteins
• Genes can respond to multiple signals and integrate response
• Regulation occurs at multiple levels in eukaryotes
Eukaryotes Prokaryotes
DNA packaged into chromatin that makes Naked DNA
it unavailable to transcription - Rely on repressors and activators
Additional RNA processing events Mature mRNA produced immediately
Transcription in nucleus and translation in Transcription and translation occurring
cytoplasm simultaneously in the cytoplasm
Regulation of transcription by the interaction of trans regulatory elements and cis
regulatory elements is the same for both eukaryotes and prokaryotes
REGULATION AT MANY LEVELS
1. Chromatin structure
2. Transcription
3. RNA processing
4. mRNA stability
5. Translation
6. Post translational modifications
1. CHROMATIN STRUCTURE
o DNA (146 base pairs) is complexed with histone octamer (2 copies of each
H2A, H2B, H3 AND H4) into nucleosomes
o Nucleosomes condense into chromatin
o Structure is dynamic and can be loosely packed or tightly
§ Controls accessibility of DNA for transcription
• Condensed chromatin – repressive
• Open chromatin = permissive
o Post translational modifications à recognized by chromatin remodelers to
uncondense and condense chromatin structure
§ Methylation (condenses)
§ Acetylation (decompresses)
• Eukaryotic DNA has two regulatory regions for genes
o Enhancers
§ Binding of regulatory transcription factors
, o Promotors – core and proximal
§ RNA pol binding to core promotor after general TF bind
2. TRANSCRIPTION
o 3 types of RNA pol in Eukaryotes
§ RNA pol 1
• Transcribes RNA components of ribosome
§ RNA pol 2
• Transcription of mRNA, microRNA, siRNA, snRNA
§ RNA pol 3
• Transcribes 5S rRNA and tRNA
• Primary RNA transcript produced is complementary to DNA antisense strand so that
the RNA transcript is a copy of the sense strand except that it has uracil not thymine
o Has exons and introns
o Poly A tail at 3’ end
• How does RNA pol find transcriptional start site?
o RNA pol on its own cannot recognize the start site
o Recruited by general transcription factors
§ GTF bind to core promotor (+- 30 nucleotides of transcription start site
– TSS)
o GTF recognize specific DNA motifs + histone modification patterns
o GTF bind to core promotor and recruit RNA pol to form the transcription
initiation complex (TIC)
o Transcription can then occur
• How was this discovered??
o EMSA à electrophoretic mobility shift assay
§ Tests for protein nucleic acid interaction
§ Uses concept of gel electrophoresis whereby molecules are separated
on size
• Apply current so as to cause nucleic acid to move through the
gel and be attracted to the positive electrode given its negative
charge due to the phosphate backbone
§ Add radioactive label to nucleic acid to allow for visualization in the
gel
• Label the nucleic acid not the protein
§ Uses small stretches of DNA (negative charge)
§ Mix DNA with proteins
• If no interaction à will see free nucleic acid at the bottom of the
gel (because small)
• If interaction à see a higher molecular weight band
o Protein slows movement of nucleic acid through gel
o Thickness of band corresponds to molecular weight of
protein
, • lane 6 – all 5 transcription
factors are present
o See a high number of
higher MW bands
o Adding one factor allows
you to see what factor binds
first (lanes 1-5)
§ TF2D binds first!!
o Causes a kink in the DNA
and then TF2B binds
§ Lanes 2-5 only show free
nucleic acid because they
cannot bind first
o Lane 7, all other factors
except TF2D added and only
the free nucleic acid is
present
§ Therefore, TF2D is
essential!
Result/Conclusion à no
direct binding of RNA pol
without transcription factors!!
• TF2D and TF2B recognize specific short sequence motifs in the core promotor (core
promotor = basal promotor)
§ Do not require all but at least some of these motifs are required in
euakryotes
§ Function = recruitment of GTF to allow RNA pol to bind and for
transcription to occur
§ Mutations would be lethal!!
o Eg. TATA/Pribnow box recognized by TF2D subunits
o Eg. TF2B recognition element
o Eg. Initiator
o Eg. Downstream promotor element
• Binding of GTF leads to kinks in DNA (bending) that are essential for transcription
o Caused by TF2D (contains TBP – Tata box binding protein)
• Transcriptional regulatory regions
o Enhancer – bound by regulatory transcription factors
o Proximal promotor – bound by regulatory transcription factors
o Core promotor – bound by general transcription factors (required for
transcription to occur)
*regulatory transcription factors determine whether or not transcription DOES occur
*different nuclei have different complements of regulatory TFs
*complement of regulatory TFs vary over time and space – allows for dynamic regulation!!
• Different genes have different binding sites
• Mis-expression of regulatory TFs can have drastic effects
