Protein synthesis and DNA mutations

Why Adapters Are Needed

• mRNA is not a physical template for amino acids

• Adapters = tRNAs, link codon (mRNA) to amino acid

tRNA: The Adapter Molecule

• ~80 nucleotides long

• Single-stranded, folds into a cloverleaf → L-shaped 3D structure

• Anticodon: Base-pairs with mRNA codon

• 3' OH: Attachment site for a specific amino acid

• Each tRNA is specific for one amino acid, based on its anticodon

  • E.g., tRNA with UAC anticodon binds AUG codon → carries methionine

Aminoacyl-tRNA Synthetases

• Enzymes that attach correct amino acid to its corresponding tRNA

• One synthetase for each of the 20 amino acids

• Recognize:

  • The correct amino acid

  • The matching tRNA

  • ATP

2 step attachment

• Amino acid + ATP → aminoacyl-AMP

• Amino acid transferred to tRNA → aminoacyl-tRNA

Wobble Base Pairing

• 61 codons, but only 40–45 tRNAs

• Wobble pairing allows flexible base pairing at the 3rd codon base

  • e.g., Inosine in tRNA can pair with U, C, or A

Ribosome structure

• Made of rRNA + proteins

• Two subunits:

  • Large (has exit tunnel and peptidyl transferase activity)

  • Small

3 ribosomal tRNA binding sites

• A-site: Aminoacyl-tRNA entry

• P-site: Peptidyl-tRNA holds growing chain

• E-site: Exit site

Translation stages

1. Initiation

2. Elongation cycle

3. Termination

Initiation

• Small subunit binds mRNA near 5’ cap

• Initiator tRNA (carrying methionine) binds AUG in P-site

• Large subunit joins

• Requires GTP + initiation factors

Elongation cycle

• Codon recognition at A-site (GTP used)

• Peptide bond formation (N to C terminus)

  • Catalyzed by peptidyl transferase (a ribozyme in rRNA)

• Translocation:

  • Ribosome shifts by 1 codon

  • tRNA in P-site ejected

  • Growing chain moves to P-site

  • Requires GTP hydrolysis

Termination

• Stop codon (UAA, UAG, UGA) enters A-site

• No tRNA matches → release factor binds

• Water added to polypeptide → released from P-site tRNA

• Ribosome dissociates (uses 2 GTPs)

Polysomes (Polyribosomes)

Multiple ribosomes translating the same mRNA simultaneously

Eukaryotic translation

• transcription/translation separated in nucleus

• proteins trafficked to locations

• mRNA transport from nucleus to cytoplasm

Prokaryotic translation

• Processes coupled

• no organelles

• no mRNA transport

Protein Targeting: ER Secretion Pathway

• Signal peptide made → bound by SRP

• SRP binds ER receptor

• Ribosome docks → polypeptide threaded into ER

• Signal peptide cleaved

• Polypeptide folds into final shape in ER

Mutation

• Permanent change in DNA

• Two Types:

  • Chromosomal: Change in gene number/position

  • Point mutations: Change in base sequence

Types of point mutations

• Substitution

• Insertion

• Deletion

Insertions/deletions often cause frameshift mutations

Somatic mutations

• Not passed onto offspring

• ~85% of cancers

Germ-line mutations

• Passed onto offspring

• inherited disorders

Spontaneous mutations

• Base tautomerism (e.g., A pairs with C)

• DNA replication errors

Induced Mutations (Mutagens)

• Base analogues (e.g., 5-bromouracil)

  • Mimic bases, mispair during replication

• Modifying agents

  • e.g., Nitrous acid → deaminates C → U → changes CG to TA

• Alkylating agents

  • e.g., Ethylation → mispairing → point mutation

• Intercalating agents

  • e.g., Ethidium bromide

  • Insert between bases → distort DNA → frameshifts

Ionising Radiation (X-rays)

• Directly damages DNA or generates free radicals

• Causes double-strand breaks

Ultraviolet (UV) Radiation

• Forms pyrimidine dimers

• Blocks replication → gaps

• Fixed by nucleotide excision repair (NER)

Ames Test

• Detects if a substance is mutagenic

• Uses mutant bacteria that can't synthesize histidine

• Reversion to growth = mutation occurred