Gene Expression and Regulation in Eukaryotes Lecture

Learning Objectives

• Explain the central dogma of molecular biology and trace information flow DNA \rightarrow RNA \rightarrow \text{Protein}.

• Identify every regulatory checkpoint that can modulate the amount, timing or activity of a gene product.

• Describe the molecular mechanisms underlying those checkpoints (chromatin modifications, transcription factors, RNA processing, translation, post-translational events, degradation).

• Compare prokaryotic versus eukaryotic strategies for doing the above.

Central Dogma of Molecular Biology

• Three irreversible* information transfers under normal physiology:

  • 1. DNA replication — semiconservative copying inside the nucleus, catalysed by DNA polymerases, always synthesised 5' \rightarrow 3'.

  • 2. Transcription — DNA template used by RNA polymerase to produce a complementary RNA; primary transcript must be processed to become mature mRNA.

  • 3. Translation — ribosome reads mRNA (cytoplasm/ER) and polymerises amino acids into polypeptide.

• *Exceptions (reverse transcriptase, RNA-dependent RNA polymerase, prions) exist but are beyond current lecture scope.

Why Regulate Gene Expression?

• All somatic cells share an identical genome yet perform specialised roles (neuron vs. myocyte vs. hepatocyte).

• Regulation allows:

  • Cellular differentiation during development

  • Adaptive responses to environmental/physiological cues

  • Energy efficiency (only needed proteins made)

• Dysregulation ➜ disease (e.g., cancer, metabolic disorders, neurodegeneration).

Master List of Regulatory Checkpoints

1. Chromatin modification (DNA level)

2. Transcription initiation

3. Co-/Post-transcriptional processing & mRNA stability

4. mRNA export & localisation

5. Translation initiation/elongation

6. Post-translational processing & chemical modification

7. Protein degradation

Chromatin Architecture & DNA-Level Control

• Chromatin = DNA + histone octamer (2×H2A, 2×H2B, 2×H3, 2×H4); linker histone H1 seals nucleosome.

• Nucleosome diameter ≈ 10\,\text{nm}; beads-on-a-string compacts into higher-order fibres.

Euchromatin vs. Heterochromatin

Histone Tail Modifications

1. Acetylation (HATs)

   • Adds \text{-COCH}_3 to lysine → neutralises positive charge → weakens DNA binding → chromatin opens.

   • Reversed by HDACs (histone de-acetylases).

2. Methylation (HMTs)

   • Adds \text{CH}_3 to Lys/Arg; does not change charge.

   • Can signal repression (H3K9me3) or activation (H3K4me3) depending on residue + degree (mono/di/tri).

3. Other marks (phosphorylation, ubiquitination, SUMOylation) create the "histone code"↔recruit or repel remodelers.

DNA Methylation (Epigenetics)

• CpG islands (promoter-proximal CG repeats) methylated by DNMTs.

• 5-methylcytosine blocks transcription factor (TF) binding → gene silencing.

• Stable through mitosis ⇒ mechanism for X-chromosome inactivation, genomic imprinting, cellular identity.

Transcriptional Regulation

• Core promoter: TATA box, initiator, +1 start site ⇒ minimal machinery.

• Proximal control elements: immediately upstream, bind specific TFs.

• Enhancers (distal elements): \pm10^3–10^6 bp away; orientation-independent.

Step-wise Assembly

1. Activator proteins bind enhancer.

2. DNA-bending proteins loop enhancer to promoter.

3. Co-activators/mediators + general TFs (TFIID with TBP, TFIIA/B/E/F/H etc.) form pre-initiation complex.

4. RNA polymerase II recruited, promoter melted, transcription starts.

Experimental note: ChIP-seq maps TF/histone mark occupancy genome-wide.

Post-Transcriptional Regulation

mRNA Processing in Nucleus

• 5′ Cap (7-methyl-guanosine via 5'!!\text{—}5' triphosphate linkage)

  1. Aids nuclear export

  2. Shields from 5'→3' exonucleases

  3. Promotes ribosome scanning/attachment

  4. Signals first intron removal

• 3′ Poly-A tail (~50–250 A residues)

  1. Enhances export

  2. Protects 3' end

  3. Cooperates with cap-binding proteins to form closed-loop during translation

• Splicing

  • Spliceosome (snRNPs + proteins) recognises GU-AG intron borders; lariat intermediate.

  • Alternative splicing → multiple isoforms from one gene → transcriptome/proteome diversity.
    Example: Tropomyosin gene produces tissue-specific variants.

mRNA Stability & Degradation

• Half-life ranges from hours (cytokine mRNAs) to days (globins).

• AU-rich elements (AREs) in 3' UTR recruit decay machinery.

• Deadenylation-dependent decay → decapping → exonucleolytic digestion.

Regulatory Non-coding RNAs

• miRNA (~22 nt): imperfect pairing → translational repression or mRNA decay (RISC complex).

• siRNA (~20 nt): perfect pairing → mRNA cleavage.

• lncRNA (>200 nt): diverse; scaffold, guide, decoy or enhancer RNA roles.

mRNA Export & Cytoplasmic Fate

1. Mature mRNP recognised by nuclear pore complex → energy-dependent export.

2. Localisation signals (zip-codes) target transcripts to neurites, poles, ER surface etc.

3. Translation begins; can be globally slowed during stress (eIF2-alpha phosphorylation).

Post-Translational Regulation

Protein Processing

• Proteolytic cleavage: pre-proinsulin ➜ proinsulin ➜ insulin.

• Chemical modifications introduce new functionalities or control localisation:

  • Phosphorylation (\text{Ser/Thr/Tyr}) — on/off switches, signalling cascades.

  • Glycosylation — folding, secretion, cell–cell recognition.

  • Lipidation, acetylation, methylation, SUMOylation.

Protein Degradation – Ubiquitin–Proteasome System (UPS)

1. E1 (activating), E2 (conjugating), E3 (ligase) enzymes attach poly-ubiquitin chain (>4 Ub) to lysine of target.

2. 26S proteasome recognises the "death tag", unfolds and chops protein into 7–9 aa peptides.

3. Provides quality control; regulates cyclins, transcription factors, misfolded proteins.

Lab methods: Western blot (targeted), Mass-spectrometry proteomics (global).

Prokaryotic vs. Eukaryotic Gene Expression — Similarities

• Both use transcription factors (activators/repressors).

• Regulation can occur at transcription, translation, and post-translation levels.

Key Differences

Implication: complexity & multilayered regulation scale with organismal complexity.

Ethical, Philosophical & Practical Implications

• Epigenetic inheritance challenges notion that DNA sequence alone dictates phenotype; environment (diet, stress, toxins) can leave methylation/acetylation marks passed to progeny.

• Targeting chromatin modifiers (HDAC inhibitors, DNMT blockers) forms basis of anti-cancer drugs.

• RNA-based therapeutics (siRNA, miRNA mimics/antagomirs) enable gene-specific silencing without genome edits.

• CRISPR-dCas9 fused to epigenetic enzymes allows programmable activation/repression — raises gene-editing ethics.

Key Numbers & Equations Recap

• DNA/RNA synthesis direction: 5' \rightarrow 3'.

• Ribosome translates mRNA\;\text{triplets} \;\Rightarrow\; 20\;\text{amino acids}.

• Ubiquitin size: 76\;\text{aa}.

• Proteasome peptide output: 7\text{–}9\;\text{aa} fragments.

• mRNA poly-A tail length: \approx 50\text{–}250 adenines.

Connections to Prior Knowledge & Real-World Relevance

• Builds on last lecture’s coverage of DNA structure & replication.

• Chromatin marks analogous to "software" overlay on genomic "hardware".

• Alternative splicing parallels modular code libraries—one gene, many outcomes.

• UPS resembles cellular "recycling centre" ensuring proteostasis, akin to waste management in cities.

• Biomedical assays (ChIP-seq, RNA-seq, mass-spec) translate these concepts into measurable data.

Self-Check Questions

1. Name three histone tail modifications and predict their effect on transcription.

2. Why does inhibiting HDACs potentially reactivate tumour-suppressor genes?

3. Explain how alternative splicing can allow a single gene to contribute to tissue specificity.

4. Contrast miRNA and siRNA mechanisms of action.

5. List two reasons eukaryotic mRNA must be capped before export.

(Answers derive directly from bullet points above.)