Genome Maintenance and Regulation

Why is regulation needed? x4

1. Efficient economy: make things in the right amount, no buildup of unnecessary proteins

2. Environmental responsiveness: respond to changes in the environment

3. Internal state responsiveness: respond to changes in cellular conditions, need to detect if there is a buildup of unnecessary proteins

4. Coordination: express groups of genes that contribute to the same process together

RNA Polymerase holoenzyme structure

2x alpha subunits - act as scaffold for holoenzyme assembly, also contain C-terminal domains (⍺CTD) which contacts UP element and activators

1x beta subunit and 1x beta prime subunit - forms crab claw and catalytic core. Beta subunit used for nucleotide addition, β’ has Mg2+ for phosphodiester bond chemistry

1x omega subunit - Assembly chaperone - helps β’ fold properly and is the smallest subunit

1x sigma factor - there are several types, attaches to core enzyme to create the holoenzyme (“whole enzyme”). This factor recognises and binds to DNA (essential for specific DNA coding)

Holoenzyme DNA recognition

• RNA polymerase needs to find a signal in the DNA to initiate transcription, sigma factor recognised specific DNA sequences

e.g. House-keeping sigma factor (sigma70) binding: recognised two sites, -10 and -35, with optimal spacer of 17bp between them BUT it can bind many variants of the -10 and -35 sites. Holoenzyme binds different binding sites less strongly, leading to different expression levels. Mutations have different effects, so that a single mutant can have a bigger effect than a double/triple

• Promoters that bind only the holoenzyme are called constitutive (always switched on)

• Sequence-dependent of sigma factor binding allows making proteins in the right amounts

Transcription initiation and elongation mechanism

1. Closed complex formation — Holoenzyme locates the promoter. UP element binds αCTDs and adds further nuance to control of expression levels.

2. Open complex formation (isomerisation) — The DNA duplex is locally melted, unwinding roughly 13 bp around the transcription start site. This creates the transcription bubble — a short stretch of single-stranded DNA exposing the template strand.

3. Abortive initiation — RNAP begins synthesising short RNA transcripts (only a few nucleotides long at a time), but repeatedly releases them without leaving the promoter.

4. Promoter escape — Once RNAP successfully synthesises a transcript >~10nt, it undergoes a conformational change, releases σ, and moves off.

5. Elongation – enzyme moves along the DNA, maintaining the bubble but also annealing the DNA back. Inside the bubble, RNA is bound to DNA, but separates due to the structure of RNAP – rudder and lid domains force RNA to peel off.

Transcription factors

Constitutive promoters are always in one state and always turned on

Second state requires a second molecule

Proteins that bind DNA

They bind specific sequences called operators, usually located near RNAP binding site

Just like RNAP, they can tolerate variation in the operator, which determines how strongly TF binds

Functionally, they can be repressor, activators, or both - depends on the binding site location

Bacterial transcription factors

Most are dimers

They bind via a defined structural motif (e.g. helix-turn-helix)

Operators are partially symmetrical due to dimeric nature

Repressor transcription factor

• Reduces RNAP binding

• Most commonly through direct hindrance or loop formation

Activator transcription factor

• Enhances RNAP binding

• Recruit RNAP usually through direct contact with αCTDs

Regulation of the Lac operon

Lac promoter (P_LAC) controls expression of an operon: set of genes expressed from the same promoter - DNA sequence where transcription begins

Three LacI operators binds to one proximal and two distal sites, then loops to provide stable repression as individually it is weak

Lac repressor (LacI) forms a dimer, is a regulatory domain that binds to allolactose from lactose metabolism, inducing conformational change and preventing DNA binding

Where does allolcatose come from?

Downstream product of lactose metabolism

lacZ - beta galactosidase: cleaves lactose into glucose and galactose - when this doesn’t work perfectly, allolactose is produced. This mechanism doesn’t always work as promoters are leaky, the extent of leakiness varies between promoters

lacY - permease: transports lactose into cell

lacA - transacetylase : detoxification enzyme, minor role

What are operons, and how does LacI differ?

Lac genes form an operon: Group of genes expressed from the same promoter, allows coordinating expression and ensuring proteins with related function are co-expressed

LacI is expressed as a single gene, its promoter is weak, efficient for proteins with independent actions, across multiple functions

Autoregulation

Autoregulation is an example of how the structure of the gene regulatory network - what transcription factors controls the expression of what genes/operons - is in itself a method of coordination and regulation

Negative auto regulation

Regulator represses to its own promoter

Fast response times and more precise expression

Positive auto regulation

Regulator activates to its own promoter

Bistability - once locked into a state, hard to go back

What two conditions does the lac operon respond to?

The presence of lactose and the absence of glucose

What is the internal signal for glucose status?

cAMP

What enzyme synthesises cAMP from ATP?

Adenylate cyclase

How does glucose affect adenylate cyclase activity and cAMP levels?

• High glucose → adenylate cyclase inhibited → low cAMP.

• Low glucose → adenylate cyclase active → high cAMP.

Role of CRP (cAMP receptor protein) in the lac operon

CRP (cAMP Receptor Protein) is the activator that detects the cAMP signal.

How is CRP activated?

CRP is inactive alone and becomes active when cAMP binds to it

How does activated CRP increase lac operon transcription?

It recruits RNA polymerase (RNAP) through direct contact with the αCTDs.

What signals does Lac operon respond to?

AND gate

Output when two conditions are true (glucose absent AND lactose present in lac operon) so transcription occurs

OR gate

Output when one of two conditions is true (x OR y present) so transcription occurs - e.g. unlike lac operon

Global control

Operons allows coordinating expression of groups of genes, but this is still a limited number

Sometimes, you need an even broader response:

• SOS response to DNA damage

• Heat shock response

• Stringent response

• Osmotic stress response

• Iron limitation response

3 common ways to achieve global regulation

Global regulators

Two-component systems

Sigma factors

Global control: Global regulators

One regulator (or a group of regulators) that act on multiple genes/operons simultaneously.

Many promoters contain binding sites - coordinate response

Allows fast responses to Environmental or Internal changes

A group of genes under the control of the same transcription factor is called a regulon

Global regulator example

e.g. CRP (cAMP receptor protein)

Many promoters contain binding sites, and it binds them in three different ways

*CRP binding site is turquoise in diagram

Global control: two-component systems

Two-component systems (TCS) take a more direct route to environmental responsiveness - doesn’t require a small molecule intermediate (ligand)

Instead, it has a dedicated protein architecture built specifically to sense an external (or internal) signal and transduce it directly into a transcriptional response, without needing a small-molecule intermediate.

Precise and coordinated response to environmental stimuli

Allows fast responses to Environmental(and less so, Internal) changes

Global control: two-component systems - Sensor histidine kinase

Membrane-embedded, with an extracellular/periplasmic sensor domain and a cytoplasmic kinase domain. Detects a specific signal (a change in osmolarity, a nutrient level, a quorum signal, etc.)

Global control: two-component systems - Autophosphorylation

Upon sensing the signal, the histidine kinase phosphorylates itself on a conserved histidine residue, using ATP

Global control: two-component systems - Phosphotransfer

The phosphoryl group is transferred from the histidine kinease’s histidine to a conserved aspartate residue on a partner protein, the response regulator.

Global control: two-component systems - Response regulator activation

Phosphorylation of the Response Regulator triggers a conformational change that activates its DNA-binding domain, letting it act as a transcription factor — activating or repressing target genes, often across a whole regulon

What defines a two component system?

Histidine → Aspartate phototransfer

• Phosphorylation of histidine and one aspartate in the response molecule receiving the phosphate

Global control: alternative signma factors

Alternative sigma factors swap out RNA polymerase's specificity subunit, redirecting the enzyme to recognize an entirely different set of promoters, genome-wide, all at once.

Most direct route to global coordinated activation

Allows fast responses to Environmental or Internal changes

Each sigma factor has its own DNA recognition sites (i.e., -10 and -35 sites are different)

E. Coli sigma factors

Global control: alternative sigma factors - Competition for limited core enzyme mechanism

Only one sigma factor can occupy core enzyme at a time, and core enzyme is present in limited amounts — so different sigma factors compete for access to core. Expression level of the sigma factors determine which promoters get transcribed.

Global control: alternative sigma factors - Anti-sigma factors

Some alternative sigma factors are always expressed, but held inactive by a dedicated anti-sigma factor that sequesters them until the appropriate signal arrives.

(e.g., σ²⁴ is held inactive by the anti-sigma factor RseA until envelope stress triggers its regulated proteolysis, releasing σ²⁴).

Global control: alternative sigma factors - Regulated synthesis/degradation

Sigma factor σ³⁸ is controlled at the level of protein stability (always generated consistently, in small amounts) but rapidly degraded under normal conditions, stabilized under stress, giving a fast on/off switch without needing new transcription of the sigma gene itself.

Global control: overlapping mechanisms

Global transcriptional control is achieve through multiple mechanisms

But they overlap in function and what they respond to: there is redundancy but also nuance

Redundancy, as several systems ensure coordinated response, when it really matters

Nuance, as different mechanisms can overlap only partially

Global control: Overlapping mechanisms SOS response example

Trigger: DNA damage or stalled replication generates single-stranded DNA (ssDNA). RecA binds ssDNA and becomes activated (RecA*).

Derepression mechanism: RecA* acts as a coprotease, stimulating LexA (the SOS master repressor) to autocleave – irreversible process. LexA normally binds SOS box in operators at >40 promoters across the genome, repressing them. As LexA is cleaved and its levels fall, repression is lost.

Temporal response/escalation programme: SOS boxes vary in LexA-binding affinity. Weak-affinity operators derepress first, so when DNA damage is low → early genes are expressed. Higher-affinity operators need more extensive LexA depletion → late genes only derepress under severe damage.

1. 2. 3. Order of escalation:

Enhanced DNA Repair - turn on genes to repair

Cell division arrest - buys time for repair, prevents faulty cells from being made

Error-prone translesion - damaging, mutagenic last resort that ignores the lesion but loses DNA code

Turning off: once damage is repaired, ssDNA disappears, RecA* deactivates, LexA is no longer cleaved, and LexA (itself under negative autoregulatory control) reaccumulates and re-represses the regulon.

Transcriptional termination

Transcription is controlled not only at the start, but at the end as well

RNA polymerase has a low likelihood of spontaneous drop-off (stopping transcription)

But genome has ways of encoding stop signs: Intrinsic and Rho-dependent termination

Intrinsic termination

Intrinsic termination exploits the difference between DNA and RNA

Specifically, RNA is single stranded - it has exposed residues so can bind/fold onto itself

Two sequence elements:

1. GC-rich inverted repeat, which, once transcribed into RNA

2. Immediately following the hairpin, a run of ~7-8 U residues in the RNA

Intrinsic termination Mechanism

• As RNAP transcribes, RNA folds into the hairpin inside the enzyme's RNA exit channel — direct physical disruption.

• Hairpin's formation mechanically slows down and destabilises the elongation complex.

• The U-tract makes dissociation easier: an RNA:DNA hybrid made of rU:dA base pairs is the weakest of all possible RNA:DNA hybrids

Rho-dependent Termination

Intrinsic termination is self-reliant; Rho-dependent require a protein, Rho

Rho is a hexameric ring-shaped ATPase, a helicase-translocase.

Two key activities of Rho:

• RNA binding — recognizes and loads onto rut sites (Rho utilization sites) sequences on the nascent RNA

• Typically 70-80 nt long, located upstream of the termination point.

• Rut sites have no strict consensus sequence — they're defined by being cytosine-rich, largely unstructured

• ATP-dependent translocation — once loaded onto the rut site, Rho uses ATP hydrolysis to translocate along the RNA in the 5′→3′ direction, chasing after the transcribing RNAP.

Rho-dependent Termination Mechanism

• Rho loads onto the nascent RNA at the rut site and begins translocating along it, 5′→3′, powered by ATP hydrolysis.

• If RNAP is transcribing at full speed, Rho typically can't catch up. But if RNAP pauses — which happens naturally at certain sequences, and is also promoted by specific pause sites near termination regions — Rho has time to catch up to the elongation complex.

• Once Rho reaches RNAP, it unwinds the RNA:DNA hybrid within the enzyme (using its helicase activity), physically destabilizing the elongation complex and causing release of RNAP, RNA, and DNA

Why is it important to have both intrinsic and rho dependent intrinsic termination?

Allows different levels of control:

Intrinsic is fast and contributes to genomic regulation by indicating transcript ends;

Rho-dependent is quality control by stopping RNAP when it transcribes what it shouldn’t.

Termination as form of transcriptional control

Not all terminators are the same - their efficiency can vary

Inefficient terminator = transcriptional read-through - terminator wasn’t working when it should’ve

Read-through provides another level of control to transcription, linking adjacent operons in a ratio of expression

RNA based control

Not all transcriptional regulation happens on promoters via protein-DNA binding

Regulation also happens after the transcript starts being made, but still during transcription. This is possible because nascent RNA can be identified and bound to

RNA-based control is different from post-transcriptional and translational regulation, which occur after transcript is fully made.

Two major mechanisms in prokaryotes: attenuation and riboswitches

Attenuation

Exploits a unique feature of bacteria - transcription-coupled translation.

Because bacteria have no nucleus, ribosomes can attach to nascent RNA as it is being transcribed and translate it on the go

Attenuation uses the position of the translating ribosome as real-time sensor of amino acid availability

Key idea: If amino acid level is high, translation is fast. If amino acid level is low, translation is slow

Allows responding to Internal signals

Attenuation: trp operon example

trp operon (tryptophan biosynthesis genes) - you want to

make amino acid synthesis genes when amino acid is low!

• Operon has upstream leader sequence, containing two consecutive tryptophan codons.

• Leader region can fold into one of two alternative RNA secondary structures, depending on ribosome position: a terminator hairpin or an antiterminator hairpin (allows RNAP to continue transcribing).

• High tryptophan means ribosome translates through the Trp codons quickly, its position favors the terminator hairpin forming → transcription stops → no wasteful synthesis of enzymes for an amino acid the cell already has.

• Low tryptophan leads to ribosome stalling, favouring the antiterminator hairpin → RNAP reads through → structural genes get transcribed → more Trp gets made.

Riboswitches

Using the nascent RNA transcript itself as the regulatory sensor, but no ribosome involvement

Instead, the mRNA directly binds a small-molecule ligand and changes conformation in response

Allows responding to Environmental and Internal signals

Riboswitches Structure

All riboswitches have the same basic architecture, located in the 5′ untranslated region of the mRNA, upstream of the operon it controls:

1.Aptamer domain — a highly structured RNA region that folds into a specific three-dimensional shape capable of binding a target ligand with high affinity and specificity;

2.Expression platform —adjacent, overlapping region that physically implements the regulatory decision. It can fold into one of two alternative structures, and which one forms depends on whether the ligand is bound.

Riboswitch mechanism

Controls formation of intrinsic terminators to achieve control:

• Ligand absent: aptamer domain remains unbound/flexible, the expression platform folds into an antiterminator structure, RNAP reads through, downstream genes are transcribed.

• Ligand bound: binding stabilizes a specific aptamer conformation, which sequesters nucleotides that would otherwise form the antiterminator — forcing the expression platform instead into a terminator hairpin causing premature termination - genes are not transcribed.

• Riboswitches overwhelmingly control genes involved in synthesizing or transporting the ligand they sense — negative loop (product senses itself, shuts down its own further production)

Patterns of gene expression during normal development

Normal development:

Begins at fertilization – the zygote is totiopotent

During development – stem cells become lineage restricted and acquire: pluripotent, then multipotent and eventually unipotent capabilities

This process correlates with their ability to express proteins of a specific cell lineage

The human body has ~250 distinct differentiated cell lineages

Differentiated cells are defined by the functions they perform – which in turn are determined by the specialised proteins that they are programmed to express

Refer to technologies for monitoring patterns of gene expression such as:

• Southern blots

• PCR (i.e. RT-PCR)

• Microarrays

• RNA seq S

Microarray analysis

Prepared and labelled with fluorescent tags:

- e.g. green probe (B-cell) and red probe (T-cell)

Probes are hybridised to DNA target sequences bound in a know pattern (i.e. grid-based) on the microarray surface

After hybridisation, samples are washed and fluorescence indicates bound probe quantity

Expression of specific loci is defined and loci clustered to monitor cell-type specific (a) or condition specific (b) patterns of expression

RNA seq (Illumina HiSeq technology) provides a state-of-the-art alternative which delivers improved quantification and can monitor all classes of RNA

Eukaryotic gene expression

• Chromatin Remodeling (Chromatin – Substrate):

  • Genomic DNA is packaged around octameric histone cores into nucleosomes, forming compact chromatin fibers.

  • Chromatin Relaxation: Epigenetic modifications (such as histone acetylation) relax condensed chromatin into accessible euchromatin so transcription machinery can access the gene template.

• Transcription and RNA Processing (Pre-mRNA):

  • RNA Polymerase II uses the exposed DNA template strand to transcribe precursor messenger RNA (pre-mRNA).

  • Co-transcriptional processing adds:

    • A 5' Cap (7-methylguanosine cap) at the 5' end.

    • A Poly-A tail (AAAA) at the 3' end.

• Splicing (mRNA):

  • Pre-mRNA contains coding regions (exons) interleaved with non-coding regions (introns).

  • The spliceosome removes the introns and ligates the exons together to form mature mRNA.

• Nuclear Export (Transport):

  • Mature, spliced mRNA bound by export factors is transported through the nuclear pore complex out of the nucleus and into the cytoplasm.

• Translation (Protein Synthesis):

  • In the cytoplasm, ribosomes bind to the 5' cap of the mRNA.

  • The ribosome reads the mRNA codons and synthesizes a growing polypeptide chain, yielding a functional protein

Class II Gene regulatory region: Promoter region

• Located immediately upstream of and around the transcription start site.

  • Upstream Sequence Element: Binding sites for regulatory transcription factors near the core promoter.

  • TATA Box (TATAAA): Consensus sequence located approximately 25 base pairs upstream of the start site.

  • Initiator (Inr) Element: Overlaps the transcription start site (+1 position)

Class II Gene regulatory region: Enhancer region

Located variable distances away (0 to 10kb or more upstream or downstream)

Assembly of RNA Polymerase II initiation complex

• TFIID = TATA Binding Protein (TBP) + TBP-Associated Factors (TAFs) (there are 10-12 TAFs)

• TBP provides the sequence-specific protein-DNA interaction (similar role in eukaryotic cells to bacterial  factor)

• TBP recruits other factors in TFIID to the promoter region and also helps with transcription stimulation

• TBP measures distance from TATA element to start site (i.e. Inr)

• TBP helps recruit TFIIA and TFIIB to determine the positioning of RNA polymerase

• TBP binds TATAA and bends the DNA at approx. 90deg - allows recruitment of other general transcription factors to the promoter

• TBP + TFIIA + TFIIB = recruit RNA polymerase II

RNA Polymerase II structure

• 10 subunits

• Catalytic activity

TFIIF

• Stabilizes RNA polymerase interaction with TBP-TFIIB

• Blocks non-specific binding of polymerase II to DNA

• Attracts TFIIE and TFIIH

TFIIE

• Attracts and regulated TFIIH

TFIIH

• Contains ATP-dependent helicase to unwind DNA

• Polymerase II C-terminal kinase that activates polymerase and releases it from the promoter

RNA Polymerase II initiation - promoter clearance

• Phosphorylation of Pol II CTD (C-Terminal Domain) by TFIIH

• TFII (D, A and B) become dispensable for elongation

• TFII (E and H-kinase) become dispensable for elongation

• Other proteins associate with TFIIH to form a repairosome

• Defects in this function are responsible for Xeroderma Pigmentosum

Stages of transcription by RNA Pol II

• Pre-initiation Complex (PIC Assembly):

  • General transcription factors (TBP/TFIID, TFIIE, TFIIH, etc.) and the Mediator complex assemble with unphosphorylated RNAPII at the promoter region (TATA box).

• Initiation and Promoter Clearance ($1 \rightarrow 2$ Transition):

  • Serine-5 Phosphorylation (Ser5-P): Upon initiation, the CTD tail of RNAPII is phosphorylated at Serine 5 (mediated by the CDK7 kinase subunit of TFIIH).

  • This phosphorylation breaks interactions with the Mediator complex, allowing RNAPII to clear the promoter.

• Promoter-Proximal Pausing and Elongation (2 → 3 Transition):

  • Pausing & Capping: Shortly after promoter clearance, RNAPII pauses (20-60bp downstream) until the 5' capping complex is recruited to cap the newly synthesized 5' end of the nascent RNA (m⁷GpppX).

  • Serine-2 Phosphorylation (Ser2-P): Transition into productive, elongation-competent synthesis requires phosphorylation at Serine 2 on the CTD by CDK9 (part of P-TEFb). Without this, transcription may abort.

Termination of transcription

• RNA polymerase II arrives at the poly-A signal (PAS) 3’-TTATTT-5’

• Adds 5’ -AAUAAA-3’ to the mRNA.

• Cleavage site is 15-30nt downstream of the PAS.

• RNA polymerase II starts adding A bases (poly-A tail) to the mRNA 3’ end.

• More efficient if there is also a U-rich upstream signal element (USE) and GU-rich downstream signal element (DSE).

Determining the DNA sequence bound by a protein when the DNA sequence is unknown

1. Generate random sequence oligonucleotides (20bp long)

2. Anneal oligonucleotides to generate all DNA combinations

3. Stick the protein to a column

4. Run the DNA over the column (protein binds to target DNA)

5. Separate bund DNA and seuqence

6. Use alignment to determine the consensus sequences (logo plot)

EMSA (electrophoretic mobility shift assay) to determine the DNA sequence bound by a protein

1. Purify the protein and add it to known DNA sequence that has been 5’- end radiolabelled

2. Run the protein-DNA on a native gel

3. DNA bound to the protein will be in a higher molecular weight

DNAse I foot printing to determine the DNA sequence bound by a protein

1. Synthesise a known DNA sequence and radiojlabel 5’ end

2. Add pure protein and then add DNAse I are a conc where it will randomly cut once per strand

3. Isolate the DNA and run on a gle. Regions where the DNA was bound to protein will have been protected (not cut) so toy get a footprint on the gel. The footprint indicated which bases were bound to protein

Modular structure of Transcription Factors

TFs contain DNA-binding domains & transcriptional regulation domains

Some have activation domains and others have repression domains

Some have domains to interact with other TFs

TFs are grouped into families on the basis of their DNA-binding domains

DNA-binding domain structures x4

Helix-Turn-Helix motif

Helix 3 binds major groove

Helices 1&2 stabilize structure

N-terminal arm contacts minor groove

Zinc finger structure

Form helical and sheet structure

Zinc fingers classes x3

Class 1 – C2-H2 – e.g. TFIIIA

Class 2 – C2-C2 – e.g. steroid receptors (note: usually simplified to C4)

Class 3 – C6 – 2xZn – e.g. GAL4

C4 zinc fingers

• A nuclear hormone receptor family

Large superfamily of ligand-dependent transcription factors/receptors

Nuclear Oestrogen Receptors are ERα and ERβ

Can bind DNA as homodimer (αα or ββ) or heterodimer (αβ)

bZip motif (Leucine Zipper) structure

• The Leucine zipper is an amphipathic helix

• Leucines at every 7th position form a hydrophobic side of the helix

• The Leucine zipper is an amphipathic helix

• Leucines at every 7th position form a hydrophobic side of the helix

• The hydrophobic helices bind to one another

• The foot of each helix is rich in basic residues

• These basic residues then bind to the DNA

• DNA-specificity depends on the bZip dimer

bHLH stricture

• Similar structure to bZip

• Helix-Loop-Helix domains interact

• The basic-regions can then bind to DNA

• They target the DNA-sequence CAnnTG (a.k.a. E-box)

• Many are heterodimers that use a common Type-1 partner

• DNA-specificity is due to the Type-2 variable partner

Activation Domains

- Activation domains share loose similarity

- Fall into a number of classes

(1) ACIDIC (Asp [D] and Glu [E] rich)

(2) GLUTAMINE RICH (Gln [Q])

(3) PROLINE RICH (Pro [P])

- Interact with GTFs/TAFs

Specific vs general transcription factors

• CLOCK-BMAL1 Complex: Form heterodimers of basic helix-loop-helix (bHLH-PAS) transcription factors that bind specifically to E-box enhancer elements (CACGTG) in target circadian genes.

• Transactivation: The glutamine-rich (polyQ) activation domain of CLOCK-BMAL1 recruits and interacts with general transcription factors (TFII complex and RNA Polymerase II at the TATA box) to stimulate gene transcription.

• General Principle: Sequence-specific TFs bind unique DNA motifs via specific DNA-binding domains (e.g., bHLH, zinc finger) and use activation domains (acidic-rich, glutamine-rich, or proline-rich) to engage basal machinery for tissue- or signal-specific regulation.

Chromatin Barrier Problem

• Nucleosome Obstacle: DNA in cells is not linear or naked; it is tightly wrapped around histone octamers to form nucleosomes (black ovals).

• Core Problem: The physical presence of nucleosomes blocks RNA Polymerase II from moving along the DNA template during elongation.

• Central Question: How does the transcriptional machinery overcome this dense chromatin barrier to allow active RNA synthesis? (This sets up chromatin remodeling and histone modification mechanisms).

Chromatin structure

DNA wrapped twice around histones - forms the nucleosome

Nucleosome structure

• 8 Core Histones (2 of each)

• ~2 Turns of DNA

• 1 Linker Histone (H1) attached on the outside

• Core tetramer of H3 and H4 bind the DNA and bend it

• The two H2A-H2B dimers then join

• The N-terminal tails are exposed and contain highly charged basic residues

• These tails are crucial for subsequent chromatin modelling

• 3 alpha helices fold domain

Histone post-transcriptional modifications x6

(1) Acetylation - of Lysine (K)

(2) Methylation - of Lysine (K) and Arginine (R)

(3) Phosphorylation - of Serine (S), Threonine (T) and Tyrosine (Y)

(4) Citrullination - of Arginine (R)

(5) Ubiquitination - of Lysine (K)

(6) Sumoylation - of Lysine (K)

Acetylation modification in lysine

H2A, H2B, H3 and H4

Methylation modification of lysine and arginine

H3 and H4

Post-translational modification of histones

• Occurs in situ (i.e. whilst histones are bound to DNA) and correlates with the functional status of the relevant genomic locus

• These changes define an epigenetic ‘histone code’ and corresponding functional ‘landscape’

• The code defines the binding potential of chromatin associated factors

Lysine acetylation

- Acetylation is a dynamic process

• Histone Acetyltransferases (HATs) add an acetyl group to Lysine - stars transcription

• Histone Deacetylases (HDACs) remove the acetyl group from Lysine - stops transcription

• Acetylation influences the structure of chromatin and TF access

• Acetylation is associated with transcriptional activation

• Acetylation = Less charge = Less cross-binding of Histone proteins = Relaxed state - DNA more accessible

Chromatin effects and transcription factors

- CLOCK-BMAL1

- bHLH-PAS transcription factors that form heterodimers

- Bind E-box enhancer sequences in circadian (i.e. biological-clock) genes

- Glutamine-rich activation domain (polyQ) interacts with the general factors

- Polymerase-II is stimulated to transcribe the gene

Histone acetylation and transcriptional activation

- CLOCK contains HAT-activity and can acetylate H3 and H4

- This opens up the core tetramer making the DNA accessible for transcription

- Hence, CLOCK relaxes the chromatin in addition to recruiting TFII+pol-II

- CLOCK keeps the basal promoter region open for TFII+Pol-II accessibility

• p300/CBP can interact with a variety of transcriptional regulators such as pCAF (a histone acetyltransferase) and TBP (which recognizes the promoter [TATAA])

• During transcription, they are all assembled at the promoter region.

• Histone acetylation by p300/CBP/pCAF and TAFII250 (also known, more simply, as TAF1) facilitates assembly of the pre-initiation complex to drive transcription

Lysine Methylation

- Methylation of Lysine can also occur

• Lysine Methyltransferases (KMTs) add a methyl group to Lysine - stops transcription

• Lysine Demethylases (KDMs) remove methyl groups from Lysine - starts transcription

- Methylation influences the structure of chromatin and TF access

- Methylation can add 1, 2 or 3 methyl groups to the NH3 + residue of lysine

- It does this by replacing a proton (H+) with CH3 each time

- Lysine hyper-methylation (me2/3) associated with transcriptional repression

Arginine Methylation

• Protein Arginine Methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (AdoMet/SAM) to arginine residues in histones (PRMT1 methylates H4R3 = transcriptional activation)

• There is a single Protein Arginine Demethylase (JMJD6) in humans! (JMJD6 demethylates H3R2 & H4R3 = transcriptional repression)

- Arginine methylation is ‘usually’ associated with transcriptional activation

• PRMT4 (CARM1) methylates H3R17 and the ‘non-histone’ protein p300/CBP

• Hence, PRMT4 methylates p300/CBP to trigger its HAT activity for transcription

How does histone modifications correlate with function?

Specific histone marks are found at predictable locations along genes – because of different functions that they perform

Active and inert genes have very different patterns of histone PT modifications.

Drives association with specific bound regulatory proteins e.g.

H3K4me3 and H3K9me3 – bind to PHD finger domain proteins – eg. ING2, BPTF - activates transcription

H3K9me2/3 and H3K27me2/3 – bind to chromodomains – eg. HP1 - activates transcription

General-Kac and H4K16ac – bind to bromodomains – eg. PCAF - represses transcription

General-Kac and H4K5acK12ac – bind to double bromodomains – eg. hTAF1 - represses transcription

Chromatin remodelling complexes

Control transitions between chromatin states

2 major classes:

• ATP-dependent chromatin remodelling histone sliding e.g. SWI/SNF, NURF

• Histone acetylase/deacetylase containing complexes e.g. SAGA, PCAF

Euchromatin - active gene

Heterochromatin - generally inactive

Remodelling allows histone sliding - reveals binding sites

Transcription factors can then recruit histone acetylases to keep the chromatin open (i.e. relaxed and accessible)

Dynamics of this process is central to gene expression

Remodelling during transcriptional elongation

• During transcription the DNA ahead of the advancing RNA Polymerase also needs to be unwound

• FACT is a heterodimeric factor that removes the H2A-H2B dimers ahead of the polymerase so that the histones disassemble to allow the Pol-II to advance and transcribe the DNA

• Once the Pol-II has passed FACT helps reassemble the histones to reform a new nucleosome that is situated further upstream (i.e. 5’) from its start position

Gene structure and transcription activation

• Transcription activation domains contact other proteins which facilitate recruitment of a functional transcription pre-initiation complex

• Three common types of activation domain – proline-[P]-rich, glutamine-[Q]-rich and acidic = glutamate-[E] and aspartate-[D]-rich

• Activators work at a distance by bringing proteins associated with key regulatory elements into a single protein super-complex at promoters

• This results in DNA looping and drives the efficiency of pre-initiation complex formation

• Locus Control Regions (LCRs) act like enhancers but work over longer distances. They contain multiple-binding-sites to allow complexes of TFs to activate multiple linked-genes

• Insulators provide boundaries for domains within euchromatin. Chromatin between two insulators is prevented from interacting with intra-chromosomal chromatin outside

• Barriers function like insulators but are more important at the boundary of heterochromatin and euchromatin. They prevent heterochromatin spreading into regions containing genes

Nuclear compartments - structure

In the mammalian nucleus chromatin folds and loops to form discrete chromosome territories

CTs are polarised so that euchromatin and heterochromatin occupy discrete zones

• Stretches of the genome can be gene-rich or gene-poor

• Different chromatin domains fold into distinct ~1 Mbp DNA foci

• Active and inactive higher-order domains are also separated

Nuclear compartments - function

RNA synthesis only occurs in certain parts of the nucleus

Molecular and cell biology approaches show that sites of mRNA synthesis are clustered within factories that typically contain many genes with 5-10 active RNA pol-II complexes

Transcriptional co-regulation in transcription factories

• 3D Spatial Clustering:

  • Upon TNF⍺ stimulation, NF-kappaB target genes loop out from different loci to cluster at a shared transcription factory enriched with RNA Polymerase II.

  • Co-localizes distant genes on the same chromosome (SAMD4A and TNFAIP2 on Chromosome 14, 50bp apart) and across different chromosomes (SLC6A5 on Chromosome 11).

• Hierarchical & Ordered Assembly:

  • Recruitment follows a set order: 1st SAMD4A → 2nd TNFAIP2 → 3rd SLC6A5.

  • TALEN Knockout: Deleting SAMD4A prevents downstream genes from co-associating, proving that pioneer locus interaction drives factory formation.

• Validation & Significance:

  • Triple RNA-FISH: Confirms physical co-localization of nascent transcripts at a single sub-nuclear spot.

  • Takeaway: 3D spatial aggregation in transcription factories enables rapid, synchronized multi-gene responses to extracellular signals.