Gene Expression: From Gene to Protein (Campbell Biology Chapter 17)

Flow of Genetic Information

• Information resides in precise nucleotide sequences of DNA.

• DNA directs synthesis of proteins that generate phenotypes; proteins link genotype ↔ phenotype.

• Gene expression comprises two stages:

  • Transcription (DNA → RNA)

  • Translation (RNA → Protein)

• Central Dogma expressed as DNA \rightarrow RNA \rightarrow Protein.

• Primary transcript = first RNA made from any gene before processing.

• In prokaryotes: transcription & translation are coupled; translation can begin before transcription ends.

• In eukaryotes: nuclear envelope separates the two; pre-mRNA undergoes RNA processing before export.

Discovery That Genes Specify Proteins

• 1902 Archibald Garrod: inherited diseases arise from inability to synthesize specific enzymes (“inborn errors of metabolism”).

• Cells run metabolic pathways of sequential enzymatic steps.

• 1940s Beadle & Tatum (Neurospora crassa bread mold):

  • X-ray–induced mutants unable to grow on minimal medium.

  • Identified 3 classes of arginine-auxotrophs; each lacked a distinct enzyme in pathway Precursor → Ornithine → Citrulline → Arginine.

  • Led to “one gene–one enzyme” hypothesis → refined to one gene–one polypeptide (because not all proteins are enzymes and some proteins contain multiple polypeptide chains).

Basic Principles of Transcription & Translation

• RNA acts as the bridge between DNA & protein.

• Transcription: DNA-dependent RNA polymerase produces messenger RNA (mRNA).

• Translation: ribosomes read mRNA codons to assemble amino-acid chain.

• Ribosomes = rRNA + proteins; serve as translation sites.

• Eukaryotic RNA transcripts are modified (5′ cap, 3′ poly-A tail, splicing) to yield mature mRNA.

The Genetic Code

• 20 amino acids vs 4 nucleotide bases → code must use combinations.

• Experiments showed triplet code; codon = 3 nucleotides → 64 possible codons.

• Properties:

  • 61 sense codons code for amino acids.

  • 3 stop codons (UAA, UAG, UGA).

  • Code is redundant (degenerate) but not ambiguous—each codon specifies only one amino acid.

  • Must be read in correct reading frame.

• Universal across life; allows gene transplantation (e.g., firefly gene in tobacco, jellyfish gene in pig).

Transcription in Detail (Concept 17.2)

Molecular Components

• RNA polymerase: separates DNA strands & links RNA nucleotides; no primer required; uses U rather than T.

• Promoter: DNA sequence where RNA polymerase binds; TATA box common in eukaryotes (≈25 bp upstream).

• Terminator (prokaryotes) / polyadenylation signal (eukaryotes) marks transcription end.

• Transcription unit: stretch of DNA transcribed into RNA.

Stages of Transcription

1. Initiation

   • Transcription factors bind promoter → recruit RNA polymerase II → form transcription initiation complex.

2. Elongation

   • RNA polymerase moves 5′→3′ on RNA, untwisting ~10–20 bp of DNA; synthesizes ≈40 nt/s in eukaryotes.

   • Multiple polymerases may transcribe a gene simultaneously.

3. Termination

   • Bacteria: polymerase stops at terminator, releases mRNA ready for translation.

   • Eukaryotes: polymerase passes AAUAAA signal; pre-mRNA cut 10–35 nt downstream; polymerase eventually releases.

RNA Processing (Concept 17.3)

Modification of Ends

• 5′ cap: modified guanine (+ 3 phosphates) added to 5′ end.

• 3′ poly-A tail: 50–250 adenines added after cleavage at AAUAAA consensus.

• Functions of cap & tail: facilitate nuclear export, protect from exonucleases, aid ribosome binding.

RNA Splicing

• Most eukaryotic genes contain introns (intervening sequences) & exons (expressed).

• Spliceosome (snRNPs + proteins) recognizes splice sites, excises intron, ligates exons.

• Ribozymes (catalytic RNAs) perform the chemistry.

• Alternative splicing: different exon combinations → multiple proteins from one gene; increases proteomic diversity.

• Exon shuffling: recombination of exons encoding distinct protein domains promotes evolution of new proteins.

Translation (Concept 17.4)

tRNA Structure & Charging

• tRNA ≈80 nt; cloverleaf 2-D, L-shaped 3-D.

• 3′ end carries amino acid (CCA). Anticodon pairs with mRNA codon.

• Aminoacyl-tRNA synthetase: 20 enzymes, each couples specific amino acid to its tRNA; reaction uses ATP:
  \text{Amino acid} + tRNA + ATP \rightarrow \text{Aminoacyl-tRNA} + AMP + 2P_i

• Wobble: flexible pairing at 3rd codon position allows one tRNA to read multiple codons.

Ribosome Anatomy

• Large + small subunits (rRNA + proteins).

• Three tRNA binding sites:

  • A (Aminoacyl): holds incoming charged tRNA.

  • P (Peptidyl): holds tRNA with growing peptide.

  • E (Exit): holds discharged tRNA before release.

• Exit tunnel in large subunit allows peptide emergence.

Stages of Translation

1. Initiation

   • Small subunit binds mRNA & initiator tRNA (Met).

   • Scans to start codon AUG; initiation factors + GTP join large subunit → translation initiation complex.

2. Elongation (repeats):

   • Codon recognition (requires GTP).

   • Peptide bond formation (peptidyl transferase in rRNA).

   • Translocation of ribosome 5′→3′ on mRNA (requires GTP).

   • Rate: in bacteria, ~15 aa/s; eukaryotes slower.

3. Termination

   • Stop codon reaches A site; release factor binds, adds H₂O → hydrolyzes bond → peptide released; complex dissociates (uses 2 GTP).

Post-Translational Events

• Polypeptide spontaneously folds into secondary/tertiary structure; chaperones may assist.

• Post-translational modifications: cleavage, phosphorylation, glycosylation, etc.

• Targeting: free ribosomes (cytosolic proteins) vs bound ribosomes (RER, secretion).

  • Signal peptide recognized by SRP → directs ribosome to ER; peptide enters lumen via translocon; signal cleaved.

• Polyribosomes (polysomes): multiple ribosomes translating one mRNA → fast/high yield.

Coupling in Bacteria vs Compartmentalization in Eukaryotes

• Bacteria: transcription & translation occur together; nascent mRNA loaded with ribosomes.

• Eukaryotes: nuclear RNA processing separates events; mRNA exported to cytoplasm for translation.

Mutations & Their Effects (Concept 17.5)

Point Mutations

• Single base-pair changes.

• Substitutions

  • Silent: no amino-acid change (redundancy).

  • Missense: wrong amino acid (e.g., sickle-cell: GAG \rightarrow GTG → Glu → Val).

  • Nonsense: codon → stop; truncates protein.

• Insertions/Deletions (indels)

  • Can cause frameshift if not multiples of 3; drastic changes, premature stop.

  • 3-nt indel removes/adds one amino acid without shift.

• If mutation harms phenotype → genetic disorder.

Sources of Mutation

• Spontaneous errors during replication, recombination, repair.

• Mutagens: physical (e.g., UV, X-ray) or chemical agents causing DNA damage.

Definitions & Evolving Concept of a Gene

• Historically: unit of heredity → DNA sequence encoding polypeptide.

• Current definition: **region of DNA expressed to produce a functional product (polypeptide *or* RNA)**.

Numerical & Statistical References

• 20 amino acids.

• 4 nucleotide bases.

• 64 codons: 61 sense + 3 stop.

• RNA polymerase elongation rate ≈ 40 nt/s (eukaryotes).

• 5′ cap added; poly-A tail length ≈ 50–250 adenines.

• Spliceosome removes introns that can be thousands of nucleotides long.

Real-World & Ethical Connections

• Universality of code enables GMO technology (e.g., fluorescent pigs, firefly-lit tobacco) → raises bioethical debates on transgenics.

• Antibiotics exploit differences between bacterial & eukaryotic ribosomes to selectively inhibit pathogens.

• Understanding mutations underpins diagnosis & treatment of genetic diseases (e.g., sickle-cell anemia).

These notes encompass all major and minor details, definitions, numerical facts, mechanisms, experimental evidence, and applications presented in the transcript, structured for comprehensive exam review.