Expression of Proteins (Translation) – Key Vocabulary

Learning Outcomes

• Discuss the principle, main steps and key molecules in protein synthesis (translation).

• Describe key post-translational modifications (PTMs) that convert a newly synthesised polypeptide into a mature, functional protein.

Central Dogma of Molecular Biology

• Proposed by Francis Crick in 1958; expanded in 1970 (Nature 227:561-563).

  • Probable information flow: DNA \rightarrow DNA (replication), DNA \rightarrow RNA (transcription), RNA \rightarrow Protein (translation).

  • Possible/rare flows: RNA \rightarrow DNA (reverse transcription), RNA \rightarrow RNA (RNA viruses), DNA \rightarrow Protein (never directly).

• 1962: Crick shared the Nobel Prize for work on nucleic-acid structure & genetics.

Protein Synthesis in Eukaryotic Cells

• "Two codes" concept

  • 4-nucleotide code (A, U, C, G) in mRNA is translated into the 20-amino-acid code of proteins.

• Translation occurs in the cytoplasm; ribosomes decode mRNA into polypeptides.

Translation: Definition, Location & Key Molecules

• Definition: Process whereby information encoded in messenger RNA directs ordered addition of amino acids to form a polypeptide.

• Cellular location: cytosol (free ribosomes) or cytosolic face of the rough ER (bound ribosomes).

• Essential components

  • mRNA (template)

  • Amino acids (building blocks)

  • Transfer RNAs (adaptors)

  • Ribosomes (rRNA + protein complexes)

  • Auxiliary factors: initiation factors (eIFs), elongation factors (eEFs), release factors (eRFs), GTP, ATP, Mg^{2+}.

Ribosomes

Structure

• Size: 2{-}4.5 MDa.

• Subunits (eukaryotes):

  • Small 40\,\text{S} + large 60\,\text{S} \Rightarrow complete 80\,\text{S} ribosome.

  • Each subunit = rRNA molecules + (~80) ribosomal proteins.

• rRNA is transcribed & processed in the nucleolus; ribosomal proteins imported from cytosol; subunits exported to cytoplasm.

• Copy number: (~10^7) ribosomes per typical mammalian cell.

Function

• Translate mRNA while moving 5' \rightarrow 3'.

• Form "polysomes" (multiple ribosomes on one mRNA) to amplify protein output.

• Provide 4 RNA-binding sites:

  • 1 mRNA tunnel

  • 3 tRNA sites: A (Aminoacyl), P (Peptidyl), E (Exit).

From RNA to Protein: Codons & Genetic Code

• Total possible codons: 4^3 = 64.

• 20 canonical amino acids coded by 61 sense codons; 3 stop codons (UAA, UAG, UGA).

• Redundancy (degeneracy): 18 amino acids have 2–6 codons each (e.g., Leu = CUU, CUC, CUA, CUG).

• Start codon: AUG \Rightarrow Methionine, establishes reading frame.

• No overlap: ribosome reads successive, non-overlapping triplets.

• Open Reading Frame (ORF): sequence from AUG to a stop codon.

Historical Milestones

• 1961: Crick et al. propose triplet codons.

• 1961: Marshall Nirenberg & Heinrich Matthaei identify UUU \rightarrow Phe.

• Later: Nirenberg, Philip Leder, Har Gobind Khorana map full code (Nobel 1968).

Transfer RNAs (tRNAs)

• Small (76-90 nt), heat-stable, soluble adaptor RNAs.

• Cloverleaf secondary/"L" tertiary shape; two critical regions:

  1. 3' CCA terminus binds specific amino acid.

  2. Anticodon loop base-pairs with complementary mRNA codon.

• Synthesised & processed in nucleus, exported to cytosol.

• Function: deliver amino acids to ribosome; enforce codon-to-amino-acid correspondence.

Four Stages of Translation (Overview)

1. Activation of amino acids ("charging" tRNAs).

2. Initiation.

3. Elongation.

4. Termination (plus ribosome recycling).

Stage 1 – Activation/Charging of Amino Acids

• Location: cytosol.

• Enzymes: 20 aminoacyl-tRNA synthetases (aaRS), each specific for one amino acid + its cognate tRNA(s).

• Two-step reaction using ATP to form high-energy ester bond:

  1. \text{AA} + ATP \rightarrow \text{AA!\mbox{-}AMP} + PP_i

  2. \text{AA!\mbox{-}AMP} + tRNA \rightarrow \text{AA!\mbox{-}tRNA} + AMP

  • Net: \text{AA} + tRNA + ATP \rightarrow \text{AA!\mbox{-}tRNA} + AMP + PP_i

• Energy later drives peptide-bond formation.

Stage 2 – Initiation (Eukaryotes)

• Events

  • Small 40\,\text{S} subunit + initiation factors bind 5' cap of mRNA; scan toward first AUG.

  • Initiator tRNA^{\text{Met}} (Met-tRNA_i^{Met}) pairs with AUG in P-site.

  • GTP hydrolysis releases factors; large 60\,\text{S} joins to form 80\,\text{S} initiation complex.

• Result: P-site occupied by Met-tRNA; A-site vacant for next aminoacyl-tRNA.

Stage 3 – Elongation

Repeated cycle:

1. Delivery: eEF1-GTP escorts next aminoacyl-tRNA to A-site; correct codon-anticodon pairing triggers GTP hydrolysis & factor release.

2. Peptide bond formation: peptidyl transferase (ribozyme activity of 28\,S rRNA) links nascent chain (in P-site) to AA in A-site.

3. Translocation: eEF2-GTP moves ribosome 3 nt toward 3' end (large subunit, then small), shifting peptidyl-tRNA from A \rightarrow P, deacylated tRNA from P \rightarrow E.

4. E-site tRNA exits; A-site open for next cycle.

• Energy: one GTP for delivery (eEF1), one GTP for translocation (eEF2) per amino acid.

Stage 4 – Termination & Ribosome Recycling

• Stop codon in A-site recognized by eukaryotic release factor eRF1 (shape mimics tRNA).

• eRF3-GTP hydrolysis triggers peptidyl-tRNA hydrolysis \rightarrow release of completed polypeptide.

• Ribosomal subunits, mRNA, and tRNA dissociate and are recycled for new rounds of translation.

Post-Translational Events

Protein Folding

• Spontaneous & chaperone-assisted acquisition of native conformation.

• Stabilising non-covalent interactions:

  • Hydrogen bonds, Van der Waals forces, ionic (electrostatic) interactions, hydrophobic effects.

• Covalent stabilisation:

  • Disulfide bridges \text{Cys}\,–S–S–\text{Cys} within a polypeptide (tertiary) or between subunits (quaternary), e.g., immunoglobulins.

Post-Translational Modifications (PTMs)

N- & C-Terminal Modifications

• N-terminal acetylation, formyl removal, myristoylation, signal-peptide cleavage.

• C-terminal amidation, glycine extension, prenylation signals, peroxisomal targeting sequences, etc.

• Influence protein stability, localisation, interaction networks (see Varland 2015; Sharma 2019; Marino 2015).

Individual Amino-Acid Residue Modifications

• Phosphorylation (Ser/Thr/Tyr) by kinases using ATP \rightarrow regulate activity, signalling cascades, transport.

• Glycosylation

  • N-linked (Asn), O-linked (Ser/Thr); affects folding, trafficking, immune recognition, protease resistance (e.g., antibody Fc glycans).

• Lipidation / Prenylation

  • Isoprenyl groups (farnesyl, geranylgeranyl) attach to Cys motifs \rightarrow membrane anchoring & signalling (e.g., Ras proteins).

• Addition of prosthetic groups

  • Heme covalently linked to His in haemoglobin.

• Ubiquitination

  • Covalent attachment of ubiquitin’s C-terminal Gly to Lys \varepsilon-amino of target protein \rightarrow marks for proteasomal degradation, endocytosis, DNA repair.

Proteolytic Processing

• Many proteins synthesised as inactive precursors (zymogens/pro-proteins) that require cleavage.

  • Example: Angiotensinogen \rightarrow Angiotensin I \rightarrow Angiotensin II (potent vasoconstrictor; ACE2 relevance in COVID-19 pathophysiology).

Summary Diagram (conceptual)

• PTMs include phosphorylation, glycosylation, lipidation, prosthetic-group addition, ubiquitination, disulfide bond formation, proteolytic cleavage—each alters structure/function, localisation or turnover.

Key Numerical & Statistical Facts

• 4\,\text{nt}^{3} = 64 possible codons vs 20 amino acids.

• 61 sense codons + 3 stop codons.

• One ribosome adds (~5{-}10) AA per second in eukaryotes; polysomes boost throughput.

• (~10^7) ribosomes per mammalian cell; ribosome mass \approx 3{-}4 MDa.

Ethical, Practical & Biomedical Connections

• PTM malfunctions linked to cancer (kinase hyper-phosphorylation), neurodegeneration (protein mis-folding/ubiquitin-proteasome defects), metabolic diseases (glycosylation disorders).

• ACE2/Angiotensin system shows how proteolytic processing intersects viral pathology (SARS-CoV-2 entry).

• Antibiotics (e.g., tetracyclines, macrolides) target bacterial ribosomal differences—illustrates applied translation biology.

Historical & Foundational Context

• Discovery of non-overlapping triplet code laid foundation for recombinant DNA & synthetic biology.

• 1968 Nobel Prize (Nirenberg, Khorana, Holley) honoured cracking of the code & tRNA structure elucidation.

Suggested Further Reading & Resources

• Crick (1970) Central Dogma review.

• Berg et al. (2021) Transfer RNA diversity.

• Varland et al. (2015) & Sharma et al. (2019) on terminal PTMs.

• Marino et al. (2015) positional proteomics of protein termini.

• Video animation of translation: https://www.youtube.com/watch?v=5bLEDd-PSTQ

• Interactive polysome model: https://ib.bioninja.com.au/.

Self-Test / Exam Practice Questions

• Describe the two high-energy bonds consumed for each amino acid incorporated during translation.

• Explain how wobble base-pairing contributes to codon degeneracy.

• Predict the effects of a mutation changing AUG (start) to ACG in an mRNA’s 5' region.

• List three PTMs that modulate protein localisation and give an example of each.

• Outline the steps by which ubiquitination targets a protein for degradation.