DG

Gene Expression & Bacterial Transcription

Context & Prerequisite Knowledge

  • Instructor’s opening reminders
    • Be able to describe the bacterial replicon and list every enzyme that acts there.
    • Distinguish \text{DNA polymerase III} (main replicative polymerase) from \text{DNA polymerase I} (primer removal & repair).
  • Ethical grading aside: instructor tries not to view names while entering scores to remain impartial.

Overview of Gene Expression vs. “Protein Synthesis”

  • Many people casually call gene expression “protein synthesis,” but that is incomplete because several distinct processes are involved:
    • 1️⃣ Transcription – DNA (\rightarrow) mRNA (performed by an RNA polymerase).
    • 2️⃣ Translation – mRNA (\rightarrow) polypeptide (performed by ribosomes).
    • 3️⃣ Post-translational processing – folding & cofactor addition to yield a functional protein.
  • Proper term “gene expression” emphasizes this multi-step path.

Directionality of Genetic Information & Viral Exceptions

  • Canonical flow: \text{DNA} \;\longrightarrow\; \text{RNA} (unidirectional for almost all cellular life).
  • Important exceptions (reverse transcription) — viruses that can go RNA (\rightarrow) DNA:
    • Hepatitis B virus (retro-like DNA virus).
    • Human Immunodeficiency Virus (HIV).
  • These exceptions create chronic, hard-to-cure infections.

Genetic Code Fundamentals

  • mRNA is read in triplets (codons); each triplet specifies a single amino acid.
  • Code is universal:
    • Example: codon \text{AUG} always (\Rightarrow) methionine in any organism.
    • “Universal” nature lets researchers swap genes across species and still obtain the same proteins.

Gene Architecture (Bacteria model)

  • Gene is divided into two positional domains referenced to the transcription start site (TSS).
    • Downstream / Coding Region
    • Extends from the first transcribed base (TSS) → end of gene.
    • Coordinates: +1, +2, +3, \dots
    • Upstream / Promoter Region
    • Lies immediately 5′ to the TSS; length ≈ 50!–!200 bp.
    • Coordinates: -1, -2, -3, \dots (no 0 exists).
    • Acts as the regulatory region controlling ON/OFF status.

Promoter Consensus Sequences

  • Two short, highly conserved subsequences recognized by RNA polymerase holo-enzyme:
    • -35 box: six bp, sequence can tolerate some A/T⇆G/C substitutions.
    • -10 box (formerly “Pribnow box,” now humorously called the TATA box):
    • Exact motif: TATAAT.
    • Absolutely invariant; any mutation here can abolish transcription.
  • Spatial relation: \text{(−35 box)}\;\;15!\text{–}!20\;\text{bp gap}\;\;\text{(−10 box)} → TSS.

Transcription – Initiation

  • Sequence of events
    • 1. Topoisomerase eases supercoils ahead of the gene.
    • 2. Helicase separates the two DNA strands within the promoter.
    • 3. Sigma (σ) factor binds simultaneously to the -35 and -10 boxes.
    • Described as “magnetically” attaching.
    • Licensing: σ binding is an absolute prerequisite; without σ, the core RNA polymerase cannot dock.
    • E. coli alone carries ≈ 7 different σ factors for various environmental conditions.
    • 4. RNA polymerase holo-enzyme sits on top of σ, slides to the +1 base, and catalyzes RNA synthesis.
  • No primer is required (contrast with DNA replication). σ performs the targeting role that a primer plays for DNA polymerases.

Transcription – Elongation

  • Terminology
    • Template strand – DNA strand actually read by RNA polymerase.
    • Coding strand – DNA strand that has the same sequence as the RNA (except T (\leftrightarrow) U).
  • Example (simplified 4 bp snippet)
    • Template: 3'-A\,T\,G\,C-5'
    • Resulting mRNA: 5'-U\,A\,C\,G-3' (matches coding strand sequence).
  • Transcript grows 5′ → 3′ as polymerase moves along the template.
  • Leader (5′ UTR) concept
    • First few bases (≥ 4) of mRNA are transcribed but not translated.
    • Critical for guiding mRNA to the ribosome and proper cellular localization.

Transcription – Termination

Two alternative, mutually exclusive pathways.

1. Extrinsic (Rho-dependent) Termination

  • Separate gene elsewhere codes the Rho (ρ) protein.
  • Steps
    • Rho recognizes a specific rut (Rho utilization) sequence on the emerging mRNA.
    • Binds, then uses ATP to translocate toward the RNA polymerase.
    • Bike metaphor: polymerase is like a bicycle needing forward momentum.
    • Rho acts as a stick in the spokes → stalls polymerase → polymerase “falls off” DNA → transcription stops.
  • Called extrinsic because termination requires an external protein factor.

2. Intrinsic (Rho-independent) Termination (GC Stem-Loop)

  • DNA template encodes a stretch of GC-rich palindromic sequence followed by poly-A.
  • When transcribed, RNA folds back on itself forming a hairpin (stem-loop) via G≡C pairing.
  • Hairpin acts as a physical anchor; polymerase stalls and dissociates.
  • No additional factors required → intrinsic termination.

Post-termination Clean-up

  • An enzyme polyadenylase adds a poly-A tail to the 3′ end of mRNA.
    • Simultaneously flattens the hairpin so the message can be translated.
  • Single-stranded binding (SSB) proteins keep RNA unfolded after tail addition.
  • Initiation resembles DNA replication but note key contrasts:
    • Topoisomerase & helicase are shared necessities.
    • Primer vs. σ factor distinction (DNA pol needs RNA primer; RNA pol needs σ).
  • Directionality principle (DNA → RNA) links to foundational “central dogma”; exceptions (reverse transcription) preview more advanced virology topics.
  • Promoter architecture parallels replication’s origin of replication; both contain core consensus motifs essential for docking of large enzymatic machines.

Practical / Ethical / Philosophical Notes

  • Instructor’s ethic: blind grading to minimize personal bias.
  • Naming conventions in molecular biology shifting from eponyms (Pribnow) to descriptive or humorous terms (TATA box) to make details more memorable.

Quick Numerical & Terminology Reference (all figures already embedded above)

  • Promoter length: 50\text{–}200 bp.
  • Coordinates: \dots,-3,-2,-1,+1,+2,+3,\dots (no 0).
  • Distance between −35 and −10 boxes: 15\text{–}20 bp.
  • σ factors in E. coli: 7.

Looking Ahead

  • Translation (ribosome structure, codon-anticodon pairing, initiation factors, etc.) will be covered in the next lecture. Stay tuned!