RNA polym*ase II Gene regulation
• RNAP I – RNA biogenesis
• RNAPII – mRNA synthesis that will be translated into proteins
• Transcription initiation at genomic regions called promoters (a DNA region at which
RNAP initiates transcription)
• GTFs and RNAP II form this complex
• DNA is packaged and bound by proteins, one aspect of regulation which is the chromatin
level which determines whether DNA is accessible or not
• (2) also needs regulatory factors to stimulate the process, also work with co-factors
o Bind to specific sequence in genome to activate genes
Gene control regions
• core promoter elements
• 5’ to 3’, left elements are upstream
• Proximal promotor – close regulatory promoter region
o Generally upstream
• In multicellular organisms, there are enhancers (distal promoters), contain binding for
o Can be up or downstream
• Come in close proximity with target promoter
Basal TIC
• Incredibly complex enzyme, but much simpler system
• Not sufficient enough to initiate transcription with a promoter
• All the general TFs need to work together to make polymerase initiate transcription
• All assemble at core promoter region to make PIC
• The bigger the letter, the more often you find
• Proteins recognise a family of sequences that are closely related, not specific sequences
only
• TATA box
• At certain genes there are specific sequences, there must be proteins that recognise this –
TATA box binding protein
• Bind TBP binds all other TFs can bind and form the PIC
o Unusual DNA binding protein (1) binds to RNA at minor groove (2) distorts DNA
when binding
o TBP looks like a saddle that sits on the DNA
• In the cell, this protein never exists alone, always in protein complexes
• TAF – TBP associated factors, also interacts with DNA
• TBP bends DNA, IIB cannot bind to DNA by itself but binds to TBP when it binds to TATA
box, interacts with DNA up and downstream of TATA box (can only bind when it bends)
• IIA
• BC – provides landing platform for all other factors to form PIC
• Transcription initiation – needs access to template strand to give RNAP access
o Transcription bubble
• As soon as RNA comes out other proteins immediately bind to process the RNA
,• Re-initiation scaffold – some genes that are transcriped over longer periods of time,
part of complex remains on promoter to make it easier to land and re-initate
transcription
How do transcription activators stimulate this process?
• S. cerevisiae regulatory process are very simple
• In multicellular organisms, regions are larger with dozens of binding sites and enhancers
with different binding sites
o Combinations of activators that regulates groups of genes
• Simplest concept is recruitment, activators land on regulatory site, comes close to site of
transcription >> interaction
o Recruit transcription machinery to promoter
o Specific combos of activate that facilitate the recruitment of transcription
machinery
• Mediator coactivator complex – involved in transcription of every gene
• Need to know how we thinking of gene re-initiation/ initiation
,Chromatin
• DNA in cells is always complex with proteins
• Histones are the primary components from chromatins > form nucleosomes
• nucleus has different areas with different densities, light and dark areas
o Euchromatin – light areas, loose chromatin, active regions
o Heterochromatin – dark areas, more or less inert, no biochemical reactions taking
place; replicated late because structure has to dissolve
• Facultative heterochromatin – can be rapidly converted into eu- and heterochromatin
• B-DNA > nucleosome array > condensed form, most highly condensed form, chromosomes
Nucleosome core particle:
• DNA is right-handed but wrapped around nucleosomes in left-handed fashion
• Dimers: H2B, H2A and H3, H4
• Protein disc referred to as histone octamer
• Term nucleosome refers to histone octamer plus DNA wrapped around, linked histone is not
part of a nucleosome complex
• histones are highly positively charged which is attracted to the negatively charged DNA
molecule
• space-filling representation: N-terminal ends of histone proteins stick out disc of nucleosome
Histone PTMs:
• N-terminals are acetylated when you have open chromatin and condensed when inactive
• HATs > acetylation > becomes inactive
o Removal of H1 protein, linker DNA
• HDACs > highly acetylated chromatin ends
• (1) modifying enzymes (2) chromatin remodelling enzymes, uses ATP to move nucleosomes
around
o Nucleosomes are not completely fixed in their position, can be moved around
• Bromodomains bind to specific histone modification
• Prevent binding of gene-specific DNA proteins (some)
• Multi-protein complex brought into
region, recruiting activity
• Results in nucleosome-free barrier
• Activators, pioneer chromatin (?)
, Trans9iption condensates – Liquid-liquid phase separation
• Membrane-less organelles are formed by liquid-liquid phase separation
• A solution consisting of the same molecule, process where one type of molecule accumulates
and forms an assembly that is separated from the surrounding solution
o “a drop within a drop”, oil and water
• at a certain concentration, just a sea of green > increasing concentration, see darker greens,
water droplets within water droplets with increasing concentration
o increasing the concentration further, droplets merge
• spontaneous process where biomolecules can form different phases
• can be stimulated by temperature, light etc
• spontaneous reactions only occur if it is energetically favourable
• entropy – a system will naturally tend towards a state that has as many states as possible?
o High entropy = high disorder
• What we saw was that entropy worked against the whole thing?
• Homotypic interaction – blue reacting with blue/ yellow with yellow
• Heterotypic interactions – blue with yellow
• Valency – the number of dynamic, weak interactions you can engage with
• Higher valency = greater network of interactions, forming complex patterns
• Weak transient interactions between multivalent proteins drive LLPS
o Multivalency is important for LLPS
• Polymers are able to undergo multivalency, no ordered structure so it can interact with many
different things at the same time
• DNA, RNA and proteins can undergo LLPS
• We know that proteins have protein domains and some
domains react with other specific domains
• Interfacial tension between the two liquid phases
• Both molecules can engage with four different
interactions
• If you have less than four, there is no phase separation
• Chains of structured regions can resemble multivalency
IDRs and IDPs
• Many proteins have regions that are not structured
• If we do not observe a structure, it does not mean that there is no structure
• Can adopt dynamically different structures at different time periods, we cannot determine a
defined structure
• Can interact with a lot of other molecules in a highly dynamic fashion
• IDP = intrinsically disordered protein
• RNAP I – RNA biogenesis
• RNAPII – mRNA synthesis that will be translated into proteins
• Transcription initiation at genomic regions called promoters (a DNA region at which
RNAP initiates transcription)
• GTFs and RNAP II form this complex
• DNA is packaged and bound by proteins, one aspect of regulation which is the chromatin
level which determines whether DNA is accessible or not
• (2) also needs regulatory factors to stimulate the process, also work with co-factors
o Bind to specific sequence in genome to activate genes
Gene control regions
• core promoter elements
• 5’ to 3’, left elements are upstream
• Proximal promotor – close regulatory promoter region
o Generally upstream
• In multicellular organisms, there are enhancers (distal promoters), contain binding for
o Can be up or downstream
• Come in close proximity with target promoter
Basal TIC
• Incredibly complex enzyme, but much simpler system
• Not sufficient enough to initiate transcription with a promoter
• All the general TFs need to work together to make polymerase initiate transcription
• All assemble at core promoter region to make PIC
• The bigger the letter, the more often you find
• Proteins recognise a family of sequences that are closely related, not specific sequences
only
• TATA box
• At certain genes there are specific sequences, there must be proteins that recognise this –
TATA box binding protein
• Bind TBP binds all other TFs can bind and form the PIC
o Unusual DNA binding protein (1) binds to RNA at minor groove (2) distorts DNA
when binding
o TBP looks like a saddle that sits on the DNA
• In the cell, this protein never exists alone, always in protein complexes
• TAF – TBP associated factors, also interacts with DNA
• TBP bends DNA, IIB cannot bind to DNA by itself but binds to TBP when it binds to TATA
box, interacts with DNA up and downstream of TATA box (can only bind when it bends)
• IIA
• BC – provides landing platform for all other factors to form PIC
• Transcription initiation – needs access to template strand to give RNAP access
o Transcription bubble
• As soon as RNA comes out other proteins immediately bind to process the RNA
,• Re-initiation scaffold – some genes that are transcriped over longer periods of time,
part of complex remains on promoter to make it easier to land and re-initate
transcription
How do transcription activators stimulate this process?
• S. cerevisiae regulatory process are very simple
• In multicellular organisms, regions are larger with dozens of binding sites and enhancers
with different binding sites
o Combinations of activators that regulates groups of genes
• Simplest concept is recruitment, activators land on regulatory site, comes close to site of
transcription >> interaction
o Recruit transcription machinery to promoter
o Specific combos of activate that facilitate the recruitment of transcription
machinery
• Mediator coactivator complex – involved in transcription of every gene
• Need to know how we thinking of gene re-initiation/ initiation
,Chromatin
• DNA in cells is always complex with proteins
• Histones are the primary components from chromatins > form nucleosomes
• nucleus has different areas with different densities, light and dark areas
o Euchromatin – light areas, loose chromatin, active regions
o Heterochromatin – dark areas, more or less inert, no biochemical reactions taking
place; replicated late because structure has to dissolve
• Facultative heterochromatin – can be rapidly converted into eu- and heterochromatin
• B-DNA > nucleosome array > condensed form, most highly condensed form, chromosomes
Nucleosome core particle:
• DNA is right-handed but wrapped around nucleosomes in left-handed fashion
• Dimers: H2B, H2A and H3, H4
• Protein disc referred to as histone octamer
• Term nucleosome refers to histone octamer plus DNA wrapped around, linked histone is not
part of a nucleosome complex
• histones are highly positively charged which is attracted to the negatively charged DNA
molecule
• space-filling representation: N-terminal ends of histone proteins stick out disc of nucleosome
Histone PTMs:
• N-terminals are acetylated when you have open chromatin and condensed when inactive
• HATs > acetylation > becomes inactive
o Removal of H1 protein, linker DNA
• HDACs > highly acetylated chromatin ends
• (1) modifying enzymes (2) chromatin remodelling enzymes, uses ATP to move nucleosomes
around
o Nucleosomes are not completely fixed in their position, can be moved around
• Bromodomains bind to specific histone modification
• Prevent binding of gene-specific DNA proteins (some)
• Multi-protein complex brought into
region, recruiting activity
• Results in nucleosome-free barrier
• Activators, pioneer chromatin (?)
, Trans9iption condensates – Liquid-liquid phase separation
• Membrane-less organelles are formed by liquid-liquid phase separation
• A solution consisting of the same molecule, process where one type of molecule accumulates
and forms an assembly that is separated from the surrounding solution
o “a drop within a drop”, oil and water
• at a certain concentration, just a sea of green > increasing concentration, see darker greens,
water droplets within water droplets with increasing concentration
o increasing the concentration further, droplets merge
• spontaneous process where biomolecules can form different phases
• can be stimulated by temperature, light etc
• spontaneous reactions only occur if it is energetically favourable
• entropy – a system will naturally tend towards a state that has as many states as possible?
o High entropy = high disorder
• What we saw was that entropy worked against the whole thing?
• Homotypic interaction – blue reacting with blue/ yellow with yellow
• Heterotypic interactions – blue with yellow
• Valency – the number of dynamic, weak interactions you can engage with
• Higher valency = greater network of interactions, forming complex patterns
• Weak transient interactions between multivalent proteins drive LLPS
o Multivalency is important for LLPS
• Polymers are able to undergo multivalency, no ordered structure so it can interact with many
different things at the same time
• DNA, RNA and proteins can undergo LLPS
• We know that proteins have protein domains and some
domains react with other specific domains
• Interfacial tension between the two liquid phases
• Both molecules can engage with four different
interactions
• If you have less than four, there is no phase separation
• Chains of structured regions can resemble multivalency
IDRs and IDPs
• Many proteins have regions that are not structured
• If we do not observe a structure, it does not mean that there is no structure
• Can adopt dynamically different structures at different time periods, we cannot determine a
defined structure
• Can interact with a lot of other molecules in a highly dynamic fashion
• IDP = intrinsically disordered protein
