Regulation of Gene Expression in Eukaryotic Cells – Vocabulary Review

Overview & Useful Resources

• Focus: Regulation of gene expression in eukaryotic cells, specifically transcription and RNA processing (Manchester Metropolitan University – Dr J. Pritchett; module: Techniques & Applications in Biomedical Science).

• Key online glossaries & molecular-biology portals:

  • https://www.genome.gov/genetics-glossary → succinct definitions (e.g. “antisense strand”, “gene”, “transcription start site”).

  • https://www.addgene.org/mol-bio-reference/promoters/ → diagrams of promoters, enhancer action, pre-initiation complex (PIC).

Learning Outcomes (Intro & Recap)

• Students will be able to:

  • Explain RNA polymerase (RNAP) function: enzymatic synthesis of RNA from DNA template.

  • Define the three stages of transcription: Initiation, Elongation, Termination.

  • Define “promoter”: cis-regulatory DNA region directing RNAP binding & TSS selection.

  • Recognise that eukaryotic RNAPs require general transcription factors (GTFs) to engage DNA.

  • Appreciate that enhancer sequences modulate transcriptional output.

  • Understand that nascent RNA is co- and post-transcriptionally processed (capping, splicing, polyadenylation).

  • Recognise that alternative RNA splicing can generate multiple mRNA isoforms from one gene.

RNA Polymerase: Types & Core Function

• General catalytic role: formation of phosphodiester bonds, extending RNA 5'\to3' by complementary base-pairing to DNA template.

• Three nuclear RNAPs in humans (plus organellar polymerases not covered):

  • RNA Pol I → synthesises \approx80–90 % of cellular rRNA (28S, 18S, 5.8S).

  • RNA Pol II → produces mRNA precursors, most snRNA, miRNA, lncRNA; clinically central because mRNAs encode protein.

  • RNA Pol III → transcribes tRNA, 5S rRNA, U6 snRNA, other small RNAs.

• All polymerases utilise Mg$^{2+}$-dependent two-metal ion catalytic mechanism (structural detail beyond scope but drives nucleophilic attack of 3'-OH on incoming NTP).

Cellular RNA Species (review)

• mRNA (messenger) → protein coding; carries open reading frame (ORF).

• rRNA (ribosomal) → scaffold & catalytic core of ribosomes.

• tRNA (transfer) → adaptor reading genetic code during translation.*

• miRNA (micro) → \sim22 nt regulators of mRNA stability & translation.

• lncRNA (long non-coding) → >200\,\text{nt} regulatory transcripts.

• Roles of rRNA & tRNA in decoding will be expanded by Dr Shalamanova in next lecture.

Canonical Stages of Transcription

1. Initiation – RNAP + GTFs assemble at promoter/TSS, form transcription bubble.

2. Elongation – RNAP traverses gene, synthesising RNA 5'\to3'.

3. Termination – RNAP disengages; nascent RNA cleaved & released; termination signals vary.

Promoter Architecture & Definition

• Promoter: DNA sequence immediately upstream (≈ −40 to +40 bp) of TSS controlling RNAP recruitment.

  • Core elements (Pol II genes): TATA box (-25), Initiator (Inr), TFIIB recognition element (BRE), Downstream promoter element (DPE).

  • Promoters integrate chromatin state, TF binding, epigenetic marks to dictate transcription frequency.

General Transcription Factors (GTFs) – Composition & Functions

• Essential for ALL eukaryotic Pol II transcription.

• Tabulated activities (Table 6-3, Molecular Biology of the Cell):

  • TFIID (TBP + TAFs): binds TATA & other core motifs; nucleates PIC.

  • TFIIB: recognises BRE; positions RNAP at +1.

  • TFIIA: stabilises TFIID–DNA complex (not universal).

  • TFIIF: escorts RNAP II; assists TFIIE/TFIIH recruitment.

  • TFIIE: recruits & regulates TFIIH.

  • TFIIH: helicase → DNA unwinding; kinase → CTD Ser5 phosphorylation, releasing RNAP into elongation.

• Net result → pre-initiation complex (PIC) assembly at promoter.

Enhancers, Chromatin Modifiers & Additional Initiation Co-factors

• Enhancers: distal DNA elements (can be >!100\,\text{kb} away); bound by activator proteins; communicate via DNA looping to promoter to stimulate PIC.

• Chromatin remodelers (SWI/SNF, ISWI, INO80, etc.) shift or evict nucleosomes, exposing promoter DNA.

• Histone modifiers (e.g., acetyltransferase Gcn5, methyltransferase Set2) deposit marks (H3K27ac, H3K4me3) associated with active transcription.

Transcription Bubble & Strand Orientation

• Bubble = \sim13–14 bp window where DNA strands are separated.

• Sense (coding) strand: sequence matches RNA (T→U).

• Antisense (template) strand: base-paired to RNA; read 3'\to5' by RNAP.

  • Visualization: DNA 3'→5' (template) / 5'→3' (coding); RNA emerges 5' end first.

Elongation: Co-factors & Chromatin Navigation

• RNAP II C-terminal domain (CTD) acts as landing pad for elongation & RNA-processing factors.

• Representative factors (Couvillion et al., 2022 list):

  • Spt4/5 (DSIF), Elf1, Dst1 (TFIIS) → influence RNAP pausing & proofreading.

  • Paf1 complex (Paf1, Leo1, Rtf1, Ctr9, Cdc73) → couples histone modification to elongation.

  • Histone chaperones (FACT, Nap1, CAF-I) → disassemble/reassemble nucleosomes.

  • Chromatin remodelers (SWI/SNF, ISWI, CHD1) → slide or eject nucleosomes ahead of RNAP.

  • CTD phosphorylation code (Ser2, Ser5, Ser7) coordinates transition from initiation → elongation → termination.

Termination: Mechanistic Models & Recent Insights

• Key facts:

  • RNA 3'-end processing (cleavage & polyadenylation) often precedes polymerase termination by \sim100$–$1000\,\text{nt}.

  • Mature mRNA receives 3' poly(A) tail; 5' end capped earlier.

• Competing models:

  1. Torpedo Model (Birch-MACH, 1998 → developed further):

  • After cleavage, 5'→3' exonuclease (e.g., Xrn2/Rat1) degrades downstream RNA, catches RNAP and destabilises elongation complex → release.

  1. Allosteric/Conformational Model: RNAP undergoes structural change upon transcribing poly(A) signal & associated pause sites, lowering processivity.

  2. Hybrid Updated Concepts (Han et al., 2023):

  • Identification of “T-tract” (thymidine-rich) pause sites + torpedo action; synergy of DNA sequence signals & exonuclease attack.

Protein-Coding Gene Structure

• Canonical features (5'→3'):

  • Promoter + TSS.

  • 5' UTR (untranslated region).

  • Exons & Introns alternately organised; number & length variable.

  • 3' UTR containing regulatory motifs (miRNA target sites, AU-rich elements).

  • Poly(A) signal (AAUAAA) followed by poly(A) tail (≈ 50$–$250\,\text{nt}) after processing.

• Transcription yields pre-mRNA containing all introns; must be processed to mature mRNA before export.

RNA Processing Steps

• 5' Capping: addition of 7-methylguanosine via 5'–5' triphosphate linkage; protects from exonucleases & aids translation initiation.

• Splicing (spliceosome-mediated): removal of introns via GU–AG rule; chemistry involves 2-step trans-esterification (branchpoint A lariat formation).

• 3' Cleavage & Polyadenylation: cleavage at CA site \sim10$–$30\,\text{nt} downstream of AAUAAA; poly(A) polymerase adds As, poly(A)-binding proteins coat tail.

• Processing is co-transcriptional; CTD Ser2 phosphorylation recruits relevant enzymes.

Alternative Splicing & Transcript Diversity

• One gene → many isoforms; combinatorial inclusion/skipping of exons.

  • Example schematic in slides: Exons 1–5 can be spliced to generate transcripts lacking exon 3 or exon 5 etc.

• Biological significance:

  • Expands proteome without increasing gene count.

  • Generates tissue-specific, developmental, or stimulus-responsive isoforms.

  • Mis-splicing → disease (e.g., spinal muscular atrophy, cancer).

Multi-Level Regulation of Eukaryotic Gene Expression (Summary Hierarchy)

1. Transcriptional Control – promoter selection, enhancer activity, chromatin accessibility.

2. RNA Processing Control – capping, splicing choices, editing.

3. RNA Transport & Localization – nuclear export through nuclear pore complex (NPC); zipcode-mediated localization in cytoplasm.

4. mRNA Stability vs Translation – miRNA/RBP binding, deadenylation, decapping; ribosome loading.*

5. Protein Activity & Degradation – PTMs, proteasomal or lysosomal turnover.
   Translation specifics covered in upcoming lecture by Dr Shalamanova.

Upcoming Related Sessions & Assessment Reminders

• Dr Shalamanova – “Translation: building proteins”, Friday 11 am.

• Dr Arora – “PCR and qPCR” (quantitative assessment of gene expression), Friday 12 pm.

• Coursework Topics:

  • Sign-up opens 18:00 (date specified); irreversible choice.

  • Submission deadline: Oct 21st 09:00.

  • Failure to self-select ⇒ topic allocated automatically.

Key Literature & Reviews Cited

• Butler & Kadonaga (2002) “The RNA polymerase II core promoter: a key component in the regulation of gene expression” Genes Dev.

• Haberle & Stark (2018) Nat Rev Mol Cell Biol – GTFs overview.

• Molecular Biology (3rd Ed, 2019) – transcription bubble illustration.

• eLife (2022) Couvillion et al. – elongation factor landscape.

• Mol Cell (2015; 2023) – Torpedo vs T-tract termination mechanisms.

Conceptual Take-Home Messages

• RNAP II cannot initiate alone; interplay of GTFs, enhancers & chromatin marks is obligatory.

• Transcription and RNA processing are spatially & temporally coupled, mediated by CTD phosphorylation cycles.

• Termination mechanisms are still under active investigation; multiple models likely co-exist.

• Alternative splicing is a major source of proteomic complexity; understanding its regulation is critical for biomedical science.

• Eukaryotic gene expression is multi-layered, permitting fine control over when, where, and how much protein is produced.

End of comprehensive lecture notes – replaces original slide set while embedding all referenced details.