Lecture 6 - Control of Gene Expression

How are transcription and translation organised in prokaryotes vs eukaryotes?

• Prokaryotes → translation is coupled with transcription

• Eukaryotes → transcription occurs in the nucleus; translation occurs in the cytoplasm

How is DNA packaged in prokaryotes vs eukaryotes?

• Prokaryotes → DNA associated with few proteins, no histones

• Eukaryotes → DNA wrapped around histones, forming complex chromatin patterns

Compare promoter complexity in prokaryotes vs eukaryotes

• Prokaryotes → relatively simple promoter sequences.

• Eukaryotes → complex transcription control sequences, including distant enhancers

How many RNA polymerases are present in prokaryotes vs eukaryotes?

• Prokaryotes → one RNA polymerase with multiple sigma factors.

• Eukaryotes → three RNA polymerases and a pre-initiation protein complex

How do prokaryotes and eukaryotes differ in introns?

• Prokaryotes → no introns

• Eukaryotes → introns present with complex splice patterns

Compare the proportion of protein-coding DNA in prokaryotes vs eukaryotes

• Prokaryotes (E. coli) → most DNA codes for proteins; 4288 protein-coding genes

• Eukaryotes (Humans) → only ~1.2% of the genome codes for proteins; ~30,000 protein-coding genes

What is the role of regulatory RNA molecules in prokaryotes vs eukaryotes?

• Prokaryotes → limited role

• Eukaryotes → expanding and significant role in gene regulation

What is the main control point for gene regulation in eukaryotes?

Transcription initiation

How does chromatin structure influence transcription?

It affects accessibility of DNA to transcription machinery; open chromatin allows transcription, condensed chromatin represses it

What enzyme synthesises RNA from a DNA template?

RNA polymerase

What is required for transcription initiation in eukaryotes?

General transcription factors and other accessory proteins

How is transcription initiation regulated?

By transcription factors that can activate or repress the formation of the initiation complex

How does post-transcriptional RNA processing regulate gene function?

Modifies RNA (capping, splicing, polyadenylation) to control stability, transport, and translation efficiency

How many RNA polymerases exist in eukaryotes and what are they?

Three: RNA polymerase I, II, and III

What does RNA polymerase I do?

Located in the nucleolus; transcribes rRNA (18S, 5.8S, 28S)

What does RNA polymerase II do?

Located in the nucleoplasm; transcribes mRNA precursors and snRNA; major control point for transcription

What does RNA polymerase III do?

Located in the nucleoplasm; transcribes tRNA and 5S rRNA

What proteins make up a nucleosome?

Eight histone proteins: two each of H2A, H2B, H3, and H4

What is the role of H1 in nucleosomes?

Acts as a linker protein, interacting with linker DNA (~60 bp) between nucleosomes

How much DNA is wrapped around the nucleosome core?

146 base pairs in 1.65 left-handed toroidal turns

What structural feature does the nucleosome create?

A dyad axis of symmetry

What happens to linker DNA when treated with micrococcal nuclease?

The linker DNA is digested, leaving nucleosome cores intact

How is chromatin remodelled dynamically?

Through processes like "worming" and "bulging," allowing flexibility and accessibility

What is "worming" in chromatin?

A sliding movement of DNA around the histone octamer, where DNA moves in a corkscrew or “worm-like” fashion

How does DNA behave during worming?

DNA unwraps slightly at one end of the nucleosome and rewraps at the other end

What drives the worming movement of nucleosomes?

ATP-dependent chromatin-remodelling complexes

What is the functional effect of nucleosome worming?

Changes the position of nucleosomes along DNA, without unwrapping the DNA entirely

From which DNA regions can nucleosomes be moved during worming?

Promoters and regulatory elements

Does DNA become unwrapped during worming?

No, DNA remains wrapped around the histone octamer; only its position shifts

What is “bulging” in chromatin?

Localised looping or bulging of DNA away from the histone surface, where part of the wrapped DNA transiently detaches

How does DNA behave during bulging?

A short stretch of DNA “pops out” while the rest remains wrapped around the nucleosome

Is bulging permanent or temporary?

It is local and temporary

What happens to the histone core during bulging?

The histone core remains in place; no nucleosome eviction occurs

What does bulging allow?

Exposes binding sites for transcription factors and DNA-binding proteins, allowing access without removing the nucleosome

What happens to arrays of nucleosomes in higher-order chromatin structure?

They condense into higher-order chromatin fibres

Why is the higher-order chromatin structure important?

It is implicated in mechanisms of chromatin repression and gene regulation

What is unknown about higher-order chromatin structure?

The exact path of DNA between nucleosomes

What are the two main theoretical models of chromatin fiber folding?

Solenoid structure (accepted theory) and zig-zag structure (theoretical possibility)

What is the third level of chromatin organisation beyond fibers?

Radial loops, where chromatin forms loops anchored to scaffolding proteins

What is special about the radial loops in chromatin?

Each loop can be independently modified, allowing localised regulation of chromatin structure and gene expression

What are histones subject to that affects chromatin structure?

Post-translational modifications

Which amino acids are targets for acetylation, methylation, phosphorylation, and ubiquitination in histones?

• Acetylation → Lysine (Lys)

• Methylation → Arginine (Arg) and Lysine (Lys)

• Phosphorylation → Serine (Ser)

• Ubiquitination → Lysine (Lys)

How do most histone modifications affect histone charge?

All modifications except methylation reduce histones’ positive charges, altering histone-DNA interaction

What is the functional significance of modified histone tails?

They interact with specific chromatin-associated non-histone proteins

What is the Histone Code Hypothesis?

Specific histone modifications evoke certain chromatin-based functions, acting sequentially or in combination to generate unique biological outcomes

What does histone acetylation do to lysine residues?

Adds acetyl groups to lysine amino acids in the histone tail, removing their positive charge

Which enzymes carry out histone acetylation?

Histone acetyltransferases (HATs)

What chromatin state and transcriptional activity is acetylation associated with?

Euchromatin and active transcription

What is histone deacetylation?

Removal of acetyl groups from lysine amino acids, adding back positive charge

Which enzymes carry out histone deacetylation?

Histone deacetylases (HDACs)

What chromatin state and transcriptional activity is deacetylation associated with?

Heterochromatin and inhibited transcription

How is histone acetylation relevant to the inactive X chromosome in females?

The inactive X lacks histone H4 acetylation, a cytogenetic marker for gene expression

How is a yeast gene typically controlled?

By multiple regulatory factors in a compact unit close to the gene

How is a human gene typically controlled?

By multiple regulatory factors located at varying distances from the transcription start site

What does the difference in regulatory sequence organisation between yeast and humans illustrate?

Increasing complexity of regulatory sequences through evolution

What is the TATA box and where is it located?

A conserved DNA sequence located ~25 bp upstream of the transcription start site

What is the role of TBP in transcription initiation?

TATA-binding protein (TBP) binds to the minor groove of the TATA box

What is the function of TFIIB?

Acts as an intermediate in the recruitment of RNA polymerase II

What is the role of TFIIF?

Assists in the recruitment of RNA polymerase II by interacting with its carboxy terminal domain (CTD)

What does TFIIE do?

Helps recruit TFIIH and modulates various activities of TFIIH

What are the key activities of TFIIH?

Helicase and kinase activities essential for transcription initiation

Which factor binds first during pre-initiation complex formation?

TFIID

What is the role of TBP in TFIID?

TBP binds the TATA box, bends the DNA, marks the promoter, and positions the transcription start site

Which factors are recruited after TFIID?

TFIIA and TFIIB

What is the function of TFIIA?

Stabilises the TBP–DNA interaction

What is the function of TFIIB?

Binds TBP and BRE elements and helps position RNA polymerase II correctly

How does RNA polymerase II arrive at the promoter?

Bound to TFIIF

What is the role of TFIIF?

Prevents non-specific DNA binding and stabilises RNA Pol II at the promoter

What is the function of TFIIE?

Binds next and creates a docking site for TFIIH

Why is TFIIH essential?

Has helicase activity to unwind DNA at the start site and kinase activity (CDK7) to phosphorylate Ser5 of Pol II CTD

What does phosphorylation of RNA Pol II CTD by TFIIH trigger?

Promoter clearance and release of RNA Pol II into elongation

What is the role of TFIIH helicase activity during transcription initiation?

Unwinds DNA at the promoter, enabling the transition from the closed to the open promoter complex

What is the role of TFIIH kinase activity during transcription initiation?

Phosphorylates RNA polymerase II CTD and may influence promoter clearance by the polymerase

What happens to RNA polymerase II after phosphorylation by TFIIH?

Phosphorylated RNA Pol II leaves the pre-initiation complex and begins RNA synthesis (elongation)

Does TFIIH remain associated with RNA polymerase II during elongation?

Yes, TFIIH stays in contact and plays a role in elongation

What happens to most general transcription factors (GTFs) during elongation?

Most GTFs detach from the core promoter

Which component of TFIID remains bound to DNA to allow re-initiation?

TFIID (possibly only TBP) remains bound to initiate a second round of transcription

Why is transcription initiation considered the critical step in gene expression?

It is the point where control over genes that impact the biochemical properties of the cell is exerted

What happens when pre-initiation complexes do not assemble efficiently?

The basal rate of transcription initiation becomes very low

How can the formation of the pre-initiation complex be activated for effective initiation?

By the action of additional proteins

What is the role of constitutive activators in gene regulation?

What is the role of constitutive activators in gene regulation?

How do regulatory activators differ from constitutive activators?

Regulatory activators target a limited number of genes and respond to external stimuli, such as hormones

How does an activator recruit RNA Pol II to the gene?

Activators recruit RNA Pol II indirectly by first recruiting transcription machinery (like the TFIID complex and/or Mediator), which then recruits RNA Pol II

What happens after activators recruit transcription machinery?

• The recruited transcription machinery, including TFIID or Mediator, helps recruit RNA Pol II to the gene

• This process is usually mediated by multiple activators bound upstream of the gene

What is the role of nucleosome modifiers in transcription?

Activators recruit nucleosome modifiers that alter chromatin near the gene, making it easier for RNA Pol II to bind

What is the Mediator complex?

An interface for transcription activators and a target for repression

What does the Mediator complex integrate?

Signals from activators and repressors bound at enhancers or promoters

How does the Mediator complex affect RNA polymerase II?

It transmits signals from activators and repressors to RNA polymerase II and the pre-initiation complex (PIC)

What is the ultimate function of the Mediator complex in transcription?

It controls whether transcription is activated, repressed, or fine-tuned

What is combinatorial control in eukaryotes?

It is the regulation of gene expression by multiple signals and regulatory proteins, contributing to the complexity and diversity of eukaryotes

How are multiple signals communicated to a gene in combinatorial control?

Each signal is transmitted by a specific regulatory protein acting on the gene

Can a single regulatory protein act on more than one gene?

Yes, regulatory protein 3 can act on both genes, but in combination with different additional regulators for each gene

What is the functional significance of combinatorial control?

It allows the same regulatory proteins to produce diverse patterns of gene expression depending on the combination of proteins present at each gene

What elements are present in both the albumin and crystallin genes?

Both have a promoter near the gene and enhancers/control elements that can be far away; these elements are the same in all cells

What determines which gene is expressed in a particular cell type?

The transcription factors (activators) available in that specific cell type

How is the albumin gene expressed in liver cells?

1. Liver-specific activators bind the albumin enhancer

2. DNA loops so enhancer contacts the promoter

3. Mediator complex and transcription machinery assemble

4. RNA polymerase II is recruited → albumin gene expressed

How is the crystallin gene expressed in lens cells?

1. Lens-specific activators bind the crystallin enhancer

2. DNA looping brings enhancer and promoter together

3. Mediator and RNA polymerase II assemble

4. Crystallin gene is expressed

What is the role of DNA looping in gene expression?

DNA looping allows distant enhancers to physically contact the promoter to facilitate assembly of the transcription machinery

Do transcription factors act alone to regulate gene expression?

No, transcription factors recruit co-regulators to assist in regulating transcription

What are co-activators, and how do they work?

Co-activators (eg. histone acetyltransferases) are recruited by transcription factors to acetylate histone tails, making DNA more accessible to the transcription machinery