DNA Transcription – Comprehensive Study Notes

Page 1: Course & Contact Information

• Topic / Block: DNA Transcription — Block 1 (Class of 2025)
• Instructor: Kanesha N. Glenn, PhD
  • Office: Biomed 150
  • Phone: 327-9857
  • E-mail: kglenn@carolinas.vcom.edu
• Institution: VCOM—Edward Via College of Osteopathic Medicine (Carolinas Campus)

Page 2: Learning Objectives

• Distinguish the roles of the three eukaryotic RNA polymerases (Pol I, II, III).
• Identify DNA-binding molecules that regulate transcription (e.g., general transcription factors, activators, repressors).
• Describe the promoter complex and its role in initiation.
• Define cis-acting DNA elements vs. trans-acting protein factors.
• Compare induction vs repression as regulatory paradigms.
• List/describe steps of eukaryotic mRNA processing: 5′ capping, intron removal, poly-A addition.
• Enumerate the major RNA types (mRNA, tRNA, rRNA, snRNP) and their distinct functions.
• Detail tRNA structure/function.
• Describe the basic parts of a typical mRNA (5′-cap, 5′-UTR, coding sequence, 3′-UTR, poly-A) and their regulatory roles.

Page 3: Definition of Transcription

• Transcription = copying of a DNA template into RNA.
• In prokaryotes: no nucleus ⇒ transcription & translation are coupled; ribosomes begin translating nascent RNA immediately.
• In eukaryotes: physical separation (nucleus vs cytoplasm) uncouples the two processes.

Page 4: Types of RNA & General Differences

• Transcription generates:
  1. mRNA – protein coding.
  2. tRNA – amino-acid adaptor.
  3. rRNA – ribosomal scaffold/catalyst.
• Key prokaryote–eukaryote contrasts
  • Number of RNA polymerases (one vs three).
  • Polycistronic (prokaryote) vs monocistronic (eukaryote) transcripts.
  • Different promoter/control sequences (Pribnow, TATA, enhancers).
  • Degree of post-transcriptional modification.
  • Compartment (nucleus vs cytoplasm).

Page 5: Mechanistic Overview of Transcription (Action of RNA Polymerase)

1. Initiation
   • Polymerase binds promoter — “closed complex.”
2. Formation of Open Complex
   • Polymerase melts DNA near start site, producing a transcription bubble.
3. First Bond Formation
   • Catalyzes phosphodiester linkage of first two rNTPs.
4. Elongation
   • Polymerase moves 3′→5′ along template, adding rNTPs 5′→3′ to RNA.
5. Termination
   • At stop site polymerase releases RNA & dissociates.

• Although the five conceptual steps are conserved, detailed protein/gene factors differ between prokaryotes & eukaryotes.

Page 6: Chemistry of RNA Polymerase Reaction

• No primer required.
• Catalyzes ester bonds between incoming rNTP and 3′-OH of growing RNA.
• Template direction: DNA read 3′→5′; RNA synthesized 5′→3′.
• Substrates: ATP, CTP, GTP, UTP; base-pairing rules:
  $T!:!A,\; C!:!G,\; G!:!C,\; A!:!U$
• Energy source: cleavage of the $\gamma$ phosphoanhydride of the rNTP (PPi released) powers bond formation.
• Lacks 3′→5′ exonuclease proofreading; error rate ≈ $1 \text{ in } 10^{5}$ bases, mitigated by kinetic proofreading/backtracking.

Page 7: RNA Polymerase Types

Prokaryotes

• One multisubunit enzyme (α₂ββ′Ω) = core.
• Association with a σ-factor ⇒ holoenzyme that recognizes promoters.
• Multiple σ-factors give gene-specific recognition; σ⁷⁰ is the house-keeping factor (≈70 kDa).

Eukaryotes

• RNA Polymerase I – rRNA (28S, 18S, 5.8S) in nucleolus.
• RNA Polymerase II – mRNA & most snRNAs; sensitive to α-amanitin.
• RNA Polymerase III – tRNA, 5S rRNA, some snRNAs.
• All share core mechanism but utilize distinct promoter architectures.

Page 8: Prokaryotic Initiation & Elongation

• σ-factor binds promoter (−35 region & Pribnow box at −10) → DNA unwinds.
• After ≈10 nt of RNA synthesized, σ is released; core continues elongation.
• Elongation persists until termination signal encountered.

Page 9: Termination Strategies

• Prokaryotes
  • ρ-independent: GC-rich inverted repeat → hairpin + U-tract destabilizes RNA–DNA hybrid.
  • ρ-dependent: ρ helicase tracks RNA, catches polymerase, unwinds hybrid.
• Eukaryotes
  • Pol I: specific termination factor.
  • Pol III: intrinsic mechanism resembling ρ-independent.
  • Pol II: transcription proceeds past poly-A signal; RNA cleaved, then degradation of downstream RNA triggers polymerase release (torpedo model).

Page 10: Prokaryotic Promoter Elements

• Promoter = DNA region controlling RNA polymerase binding/start site.
• Pribnow box (−10): $5'\text{-TATAAT-}3'$; AT-rich lowers melting energy.
• σ-factor recognizes −35 region ($\sim$TTGACA) & Pribnow; initiates strand separation.

Page 11: Eukaryotic Core Promoters

• May contain multiple modules; most famous = TATA box (≈−25): $5'\text{-TATAAA-}3'$.
• Binds TFIID (multi-protein basal factor complex).
  • TBP = TATA-binding protein (subunit of TFIID).
• Other basal/general TFs: TFIIB, TFIIE, TFIIF, TFIIH (ATP-dependent helicase/kinase).

Page 12: Assembly of the RNA Pol II Pre-Initiation Complex (PIC)

1. TBP (within TFIID) binds TATA → bends DNA.
2. TFIIA & TFIIB stabilize TBP–DNA & act as bridge.
3. RNA Pol II (with TFIIF) docks; aligned at +1.
4. TFIIE & TFIIH join; TFIIH helicase activity opens DNA; its kinase phosphorylates Pol II C-terminal domain (CTD).
5. ATP hydrolysis triggers promoter clearance → elongation.

Page 13: Proximal & Distal Control Elements / Enhancers

• Proximal elements: within ≈100–200 bp of start; sometimes grouped with “promoter.”
• Distal enhancers/silencers: can be $>10^{3}$ bp away, upstream, downstream, or inside introns.
• DNA bending (by architectural proteins) allows activator proteins at enhancers to interact with the PIC via mediator complex.
• A DNA-binding protein that increases transcription = activator; one that inhibits = repressor.

Page 14: Response Elements & Signal-Dependent Regulation

• Heat Shock Element (HSE), Glucocorticoid Response Element (GRE), Metal Response Element (MRE) are shared DNA modules.
• Binding of inducible transcription factors converts environmental signals (heat, hormone, metal) into transcriptional activation of any promoter nearby.
• Illustrates cis–trans logic: cis DNA sequence + trans protein factor.

Page 15: Gene Organization—Prokaryotes vs Eukaryotes

(a) Prokaryotic operon (e.g., E. coli trp operon)
• Single promoter → polycistronic mRNA encoding enzymes trpE-A.
• Start site for transcription upstream of 5 genes; ribosome binding sites before each ORF.
(b) Eukaryotic arrangement
• Related genes (e.g., yeast TRP1-5) located on different chromosomes.
• Coordinated expression via shared control elements rather than shared promoter.

Page 16: Anatomy of a Human Gene

• Exons: coding sequences retained in mature mRNA.
• Introns: non-coding sequences spliced out; enable alternative splicing.
• Multiple upstream & downstream control elements enhance regulatory complexity.
• Post-transcriptional additions: 5′ cap, 3′ poly-A tail.

Page 17: 5′ Capping Mechanism & Purpose

Biochemical steps (co-transcriptional):

1. Terminal triphosphate of nascent RNA loses $\gamma$ P.
2. Remaining $\beta$-phosphate attacks $\alpha$-P of GTP ⇒ 5′–5′ triphosphate bridge (unique orientation).
3. Methylation by S-adenosyl-L-methionine (SAM):
   • N7 of guanine.
   • 2′-OH of first nucleotide (sometimes 2nd) ⇒ “cap 1”/“cap 2”.
   Functions

• “Seals” 5′ end against 5′→3′ exonucleases.
• Serves as recognition signal for eIF4E during translation initiation.

Page 18: Poly-A Tail Addition & Purpose

Process

1. RNA Pol II transcribes through polyadenylation signal (AAUAAA).
2. Endonuclease complex cleaves ~10–30 nt downstream.
3. Poly(A) polymerase (PAP) adds >200 A residues using ATP, template-independent.
   Functions

• Binding site for Poly(A)-binding protein (PABP) → protects from 3′ exonucleases.
• Facilitates nuclear export & promotes translation by circularizing mRNA with 5′ cap.

Page 19: Integrated View—Cap & Tail Cooperation

• 5′ cap and 3′ poly-A tail interact via protein bridges (eIF4G, PABP) to form “closed-loop” mRNP, enhancing translational efficiency & surveillance.

Page 20: Intron Removal by Spliceosome

Consensus

• 5′ splice site: GU
• 3′ splice site: AG
• Branch-point A upstream of 3′ site.
  Components
• snRNPs (U1, U2, U4, U5, U6) + proteins = spliceosome.
  Mechanism

1. U1 binds 5′ GU; U2 base-pairs with branch-point A.
2. U4/U5/U6 recruited; U6 displaces U1; active site forms.
3. First transesterification: 2′-OH of branch A attacks 5′ splice site → lariat.
4. Second transesterification: 3′-OH of exon 1 attacks 3′ splice site → exons ligated; lariat released & debranched.

• Spliceosome cycling consumes ATP for assembly/rearrangement.

Page 21: Schematic of Splicing (Visual Slide)

• Depicts looped lariat intron; highlights positions of snRNPs and transesterification points.
• Emphasizes coordinated release of intron for degradation to mononucleotides.

Page 22: Major Eukaryotic vs Prokaryotic Differences (Summary)

• Processing: capping, poly-A, splicing only in eukaryotes.
• Polymerases: 3 vs 1.
• Gene Organization: monocistronic vs polycistronic.
• Compartmentalization: transcription in nucleus, translation in cytoplasm (euk.), vs coupled in prok.
• Promoter Complexity: enhancers, mediators, chromatin context in eukaryotes.

Page 23: Four Major RNA Classes & Abundance

1. rRNA (~80%) — catalytic/protein scaffold of ribosome.
2. tRNA (~15%) — adapter linking codon to amino acid.
3. mRNA (
4. snRNPs — spliceosome components (~100–200 nt RNA + proteins).

Page 24: tRNA Structure & Charging

• Cloverleaf secondary; L-shaped tertiary.
• 3′ end: CCA sequence; amino acid esterified to 3′-OH ribose.
• Anticodon loop confers codon specificity.
• Aminoacyl-tRNA synthetases (20 distinct) catalyze charging:
  $\text{Amino Acid} + \text{tRNA} + ATP \rightarrow \text{Aminoacyl-tRNA} + AMP + PP_i$
• Proofreading by synthetases ensures fidelity of translation.

Page 25: Key Take-Home Concepts

• Transcription = DNA-dependent RNA synthesis by RNA polymerase.
• Template (non-coding) strand read 3′→5′; RNA identical (except U for T) to coding strand.
• RNA polymerases lack primer & 3′→5′ exonuclease; error rate ~$10^{-5}$.
• Promoter sequences dictate initiation position & efficiency.
• Auxiliary elements (proximal, enhancers) modulate rate via DNA-binding proteins.
• Eukaryotic primary transcript undergoes capping, splicing, polyadenylation.
• Mature mRNA exported → translated in cytoplasm; introns provide potential for alternative splicing & proteomic diversity.

Page 26: Institutional Footer (No New Content)

• VCOM—Edward Via College of Osteopathic Medicine (Carolinas Campus).