# Lecture 27: Translation and Protein Targeting

## Biochemistry

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## Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the genetic code and its key properties (degeneracy, universality, wobble)
2. Explain the role of tRNA, aminoacyl-tRNA synthetases, and ribosomes in translation
3. Describe the three phases of translation: initiation, elongation, and termination
4. Compare prokaryotic and eukaryotic translation and identify key differences
5. Explain the mechanisms of co-translational and post-translational protein targeting
6. Describe post-translational modifications and their functional significance
7. Discuss antibiotics and toxins that target translation

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## Lecture Content

### I. The Genetic Code

The genetic code is a **triplet code** in which each codon, consisting of three consecutive nucleotides in mRNA, specifies one amino acid. There are **64 codons** in total: 61 sense codons that encode amino acids and 3 stop codons. The **start codon** is **AUG**, which encodes methionine and also serves as an internal Met codon. In prokaryotes, AUG at the start position encodes **N-formylmethionine (fMet)**. The three **stop codons** are **UAA** (ochre), **UAG** (amber), and **UGA** (opal); these do not encode amino acids but are instead recognized by release factors rather than tRNAs.

The genetic code possesses several important properties. It is **degenerate** (redundant), meaning most amino acids are encoded by more than one codon, with the exceptions of methionine and tryptophan, which have only one codon each. It is **unambiguous** because each codon specifies only one amino acid. It is **non-overlapping**, with codons read sequentially without sharing nucleotides. It is **commaless**, with no punctuation between codons and the reading frame set entirely by the start codon. Finally, it is **nearly universal**, with the same code used in almost all organisms, though minor exceptions exist in mitochondria and some protists.

The **wobble hypothesis**, proposed by Crick, explains that the third base of the codon (3' position) can form non-standard base pairs with the first base of the anticodon (5' position). This allows a single tRNA to recognize multiple codons that differ only at the third position. Inosine (I) in the anticodon wobble position can pair with U, C, or A, which explains why degeneracy is concentrated at the third codon position.

### II. Transfer RNA (tRNA) — The Adaptor Molecule

Transfer RNA is a small RNA molecule of approximately 75-90 nucleotides that adopts a characteristic **cloverleaf secondary structure** and an **L-shaped tertiary structure**. Its key structural features include the **acceptor stem**, where the 3'-CCA end provides the attachment point for the amino acid via an ester bond to the 3'-OH of the terminal adenosine. The **anticodon loop** contains the three-nucleotide anticodon that is complementary to the mRNA codon. The **D loop** contains dihydrouridine, and the **T-psi-C loop** contains thymidine, pseudouridine, and cytidine. Transfer RNAs also contain extensive **base modifications** including methylation and deamination.

#### Aminoacyl-tRNA Synthetases:

There are **20 different synthetases**, one for each amino acid. These enzymes catalyze the **charging** (aminoacylation) of tRNA in a two-step reaction. In the first step, the amino acid reacts with ATP to form aminoacyl-AMP plus pyrophosphate (activation). In the second step, the aminoacyl group is transferred from aminoacyl-AMP to the tRNA, releasing AMP. The overall reaction consumes the equivalent of 2 high-energy bonds because PPi is subsequently hydrolyzed to 2Pi, which drives the reaction forward. Some synthetases possess **proofreading** capability through an editing site that hydrolyzes incorrectly attached amino acids, ensuring the fidelity of the genetic code. Recognition of the correct tRNA involves **identity elements**, which are specific bases located primarily in the acceptor stem and anticodon.

### III. Ribosomes — The Translation Machinery

The **prokaryotic ribosome** sediments at 70S and consists of a 30S small subunit (containing 16S rRNA and 21 proteins) and a 50S large subunit (containing 23S rRNA, 5S rRNA, and 34 proteins). The **eukaryotic ribosome** sediments at 80S and consists of a 40S small subunit (containing 18S rRNA and approximately 33 proteins) and a 60S large subunit (containing 28S rRNA, 5.8S rRNA, 5S rRNA, and approximately 49 proteins).

The ribosome has three **tRNA binding sites**. The **A site** (aminoacyl) accepts the incoming aminoacyl-tRNA. The **P site** (peptidyl) holds the tRNA carrying the growing peptide chain. The **E site** (exit) holds the deacylated tRNA before it leaves the ribosome. Critically, the **peptidyl transferase** activity that catalyzes peptide bond formation resides in the **23S rRNA** (in prokaryotes) or 28S rRNA (in eukaryotes), making the ribosome a **ribozyme**.

### IV. Translation: Initiation

#### Prokaryotic Initiation:

Prokaryotic initiation involves three initiation factors: **IF-1, IF-2 (a GTPase), and IF-3**. IF-3 prevents premature association of the 30S and 50S subunits. The 30S subunit binds the mRNA at the **Shine-Dalgarno sequence** (AGGAGG, located approximately 8 nucleotides upstream of AUG), which base-pairs with the 3' end of 16S rRNA. **fMet-tRNAfMet** (the initiator tRNA carrying N-formylmethionine) is then delivered to the P site by IF-2-GTP. Finally, the 50S subunit joins, IF-2 hydrolyzes GTP, and the initiation factors are released, forming the complete 70S initiation complex.

#### Eukaryotic Initiation:

Eukaryotic initiation is more complex, involving more than 12 initiation factors (eIFs). **eIF2-GTP** delivers **Met-tRNAiMet** (the initiator tRNA that carries regular Met rather than fMet) to the 40S subunit, forming the 43S preinitiation complex. **eIF4E** binds the 5' m7G cap, while **eIF4G** (a scaffolding factor) bridges eIF4E, the poly(A)-binding protein (PABP), and the ribosome, effectively circularizing the mRNA. **eIF4A**, an RNA helicase, unwinds secondary structure in the 5' UTR.

The 43S complex then scans along the mRNA from the 5' cap in the 5' to 3' direction until it encounters the **first AUG** in a favorable context known as the **Kozak sequence** (GCC**A/G**CCAUGG). AUG recognition triggers GTP hydrolysis by eIF2, and eIF5 promotes 60S subunit joining to form the **80S initiation complex**. Eukaryotes do not use a **Shine-Dalgarno sequence**; cap-dependent scanning is the primary mechanism. An exception exists in **IRES** (internal ribosome entry sites), which allow cap-independent translation and are used by some viruses and under cellular stress conditions.

<image>A side-by-side comparison diagram of prokaryotic and eukaryotic translation initiation. Left panel (Prokaryotic): The 30S subunit is shown with the Shine-Dalgarno sequence on mRNA base-pairing with 16S rRNA. IF-2-GTP delivers fMet-tRNAfMet to the P site at the AUG start codon. The 50S subunit joins to form the 70S initiation complex. Right panel (Eukaryotic): The 43S preinitiation complex (40S subunit + eIF2-GTP-Met-tRNAiMet + other eIFs) is recruited to the 5' cap (m7G) via eIF4E and eIF4G. The complex scans along the 5' UTR until it reaches the AUG in Kozak context. GTP hydrolysis occurs, the 60S subunit joins to form the 80S initiation complex. Key differences are highlighted in boxes: Shine-Dalgarno vs. cap-scanning, fMet vs. Met, and the circularization of mRNA via eIF4G-PABP interaction with the poly(A) tail.</image>

### V. Translation: Elongation

The elongation cycle adds one amino acid per cycle and is highly conserved between prokaryotes and eukaryotes. It consists of three steps per cycle.

**Step 1: Aminoacyl-tRNA Delivery (Decoding)** — **EF-Tu-GTP** (in prokaryotes) or **eEF1A-GTP** (in eukaryotes) delivers aminoacyl-tRNA to the **A site**. Codon-anticodon recognition is verified, and correct base pairing triggers GTP hydrolysis by EF-Tu. EF-Tu-GDP is then released and recycled to EF-Tu-GTP by **EF-Ts** (a guanine nucleotide exchange factor). Incorrect tRNAs are rejected before GTP hydrolysis through a process called kinetic proofreading.

**Step 2: Peptide Bond Formation (Transpeptidation)** — The **peptidyl transferase** (23S rRNA in prokaryotes or 28S rRNA in eukaryotes, functioning as a ribozyme) catalyzes the transfer of the growing peptide from the P-site tRNA to the amino group of the A-site aminoacyl-tRNA. This forms a new peptide bond, leaving the P-site tRNA deacylated and the growing peptide now attached to the tRNA in the A site.

**Step 3: Translocation** — **EF-G-GTP** (in prokaryotes) or **eEF2-GTP** (in eukaryotes) catalyzes translocation of the ribosome by one codon (3 nucleotides) along the mRNA in the 5' to 3' direction. The deacylated tRNA moves from the P site to the E site and then exits, while the peptidyl-tRNA moves from the A site to the P site. The A site is now empty and ready for the next aminoacyl-tRNA. GTP hydrolysis by EF-G drives this translocation.

**Energy Cost per Amino Acid Added:** Each amino acid addition requires 1 ATP for aminoacyl-tRNA charging (ATP converted to AMP + PPi, equivalent to 2 high-energy bonds), 1 GTP for EF-Tu/eEF1A-mediated A-site delivery, and 1 GTP for EF-G/eEF2-mediated translocation. The **total is approximately 4 high-energy phosphate bonds per amino acid** (2 from ATP + 1 GTP + 1 GTP).

### VI. Translation: Termination

When a **stop codon** (UAA, UAG, or UGA) enters the A site, **release factors** bind instead of tRNA. In prokaryotes, **RF-1** recognizes UAA and UAG, **RF-2** recognizes UAA and UGA, and **RF-3** is a GTPase. In eukaryotes, **eRF1** recognizes all three stop codons and **eRF3** functions as the GTPase. Release factors trigger hydrolysis of the ester bond between the peptide and the P-site tRNA, allowing the completed polypeptide to be released. The ribosome is then dissociated into its subunits for reuse by the **ribosome recycling factor (RRF)** plus EF-G in prokaryotes, or ABCE1 in eukaryotes.

### VII. Polyribosomes (Polysomes)

Multiple ribosomes can translate a single mRNA simultaneously, forming a polyribosome or polysome. The first ribosome is closest to the 3' end of the mRNA (furthest along in translation), and polysomes increase the rate of protein production from a single mRNA molecule. **Free polysomes** synthesize cytoplasmic, nuclear, mitochondrial, and peroxisomal proteins, while **membrane-bound polysomes** on the rough ER synthesize secreted, membrane, and lysosomal proteins.

<image>A diagram of the translation elongation cycle. A ribosome is shown with its three tRNA binding sites (E, P, A) straddling an mRNA strand. Step 1 (Decoding): EF-Tu-GTP delivers an aminoacyl-tRNA to the A site; codon-anticodon matching is shown with correct base pairing; GTP is hydrolyzed, and EF-Tu-GDP is released (recycled to EF-Tu-GTP by EF-Ts). Step 2 (Peptide bond formation): The peptidyl transferase center (in 23S rRNA) catalyzes the transfer of the peptide chain from the P-site tRNA to the A-site amino acid, forming a new peptide bond. The P-site tRNA is now deacylated. Step 3 (Translocation): EF-G-GTP binds, GTP is hydrolyzed, the ribosome shifts one codon to the right (5'->3'). The deacylated tRNA moves to the E site and exits, the peptidyl-tRNA moves from A to P, and the A site is empty for the next cycle. The energy cost (2 GTP per elongation cycle + 2 high-energy bonds for tRNA charging) is annotated.</image>

### VIII. Protein Targeting and Sorting

#### A. Signal Hypothesis and the Secretory Pathway

Proteins destined for secretion, the plasma membrane, lysosomes, or the ER/Golgi contain an **N-terminal signal peptide** of approximately 16-30 amino acids with a hydrophobic core. Co-translational targeting to the ER proceeds through a series of ordered steps. First, the signal peptide emerges from the ribosome. Then the **Signal Recognition Particle (SRP)** binds the signal peptide and pauses translation. SRP docks with the **SRP receptor** on the ER membrane, and the ribosome is transferred to the **translocon** (the Sec61 complex, a protein-conducting channel). Translation resumes, and the growing polypeptide is threaded through the translocon into the ER lumen. **Signal peptidase** cleaves the signal peptide inside the ER lumen, and SRP and the SRP receptor are released through GTP hydrolysis by both.

#### B. Post-Translational Modifications in the ER and Golgi

**N-linked glycosylation** begins in the ER, where a preformed 14-sugar oligosaccharide (Glc3Man9GlcNAc2) is transferred from dolichol phosphate to the **Asn** residue within the sequence **Asn-X-Ser/Thr** (the sequon) by oligosaccharyltransferase. Glucose residues are trimmed in the ER as part of quality control through the calnexin/calreticulin cycle, and further trimming and modification occurs in the Golgi. **O-linked glycosylation** occurs in the Golgi, where sugars are added to Ser or Thr residues one at a time. **Disulfide bond formation** is catalyzed by **protein disulfide isomerase (PDI)** in the oxidizing environment of the ER lumen. **Protein folding** is assisted by ER chaperones including BiP/GRP78, calnexin, and calreticulin.

#### C. Protein Quality Control

The **unfolded protein response (UPR)** is activated when misfolded proteins accumulate in the ER. It reduces global translation through PERK-mediated phosphorylation of eIF2alpha, increases chaperone expression, and activates ER-associated degradation (ERAD), whereby misfolded proteins are retrotranslocated to the cytoplasm and degraded by the **proteasome** after ubiquitin tagging. A clinically important example is **I-cell disease** (mucolipidosis II), caused by deficiency of **GlcNAc phosphotransferase**, which leads to failure to add the mannose-6-phosphate (M6P) tag to lysosomal enzymes. As a result, these enzymes are secreted rather than targeted to lysosomes, causing a lysosomal storage disorder with characteristic inclusion bodies in cells.

#### D. Other Targeting Pathways

**Nuclear proteins** contain a **nuclear localization signal (NLS)** consisting of a basic amino acid sequence and are imported through nuclear pores by importins via the Ran-GTP cycle. **Mitochondrial proteins** contain an N-terminal **mitochondrial targeting sequence** (an amphipathic, positively charged helix) and are imported post-translationally through the TOM/TIM translocases. **Peroxisomal proteins** contain a **PTS** (peroxisomal targeting signal), often the C-terminal tripeptide SKL, and are imported by the Pex5 receptor.

### IX. Post-Translational Modifications (PTMs) — Summary

| Modification | Enzyme/Location | Function |
|---|---|---|
| Phosphorylation | Kinases / cytoplasm | Signal transduction, enzyme regulation |
| Glycosylation (N-linked) | ER, Golgi | Protein folding, stability, targeting |
| Glycosylation (O-linked) | Golgi | Cell signaling, mucin structure |
| Ubiquitination | Ubiquitin ligases (E1, E2, E3) / cytoplasm | Targets proteins for proteasomal degradation |
| Acetylation | Acetyltransferases | Histone regulation, protein stability |
| Methylation | Methyltransferases | Histone regulation, signaling |
| Proteolytic cleavage | Proteases | Activation of zymogens (e.g., insulin, digestive enzymes) |
| Disulfide bond formation | PDI / ER | Protein structure stabilization |
| Lipid modification (prenylation, myristoylation, palmitoylation) | Cytoplasm/ER | Membrane anchoring |
| Hydroxylation | Prolyl/lysyl hydroxylases / ER | Collagen stability (requires vitamin C) |
| Carboxylation (gamma-carboxylation) | Gamma-glutamyl carboxylase / ER | Activation of clotting factors (requires vitamin K) |

### X. Antibiotics and Toxins Targeting Translation

| Agent | Target | Effect |
|---|---|---|
| **Tetracycline** | 30S subunit (A site) | Blocks aminoacyl-tRNA binding |
| **Aminoglycosides** (gentamicin, streptomycin) | 30S subunit | Causes mRNA misreading; blocks initiation |
| **Chloramphenicol** | 50S subunit (peptidyl transferase) | Blocks peptide bond formation |
| **Macrolides** (erythromycin, azithromycin) | 50S subunit (exit tunnel) | Blocks translocation |
| **Clindamycin** | 50S subunit | Blocks translocation |
| **Linezolid** | 50S subunit | Blocks initiation complex formation |
| **Diphtheria toxin** | eEF2 (ADP-ribosylation) | Inactivates eukaryotic elongation factor 2 |
| **Pseudomonas exotoxin A** | eEF2 (ADP-ribosylation) | Same mechanism as diphtheria toxin |
| **Ricin** (from castor beans) | 28S rRNA | Depurinates a specific adenine; inactivates 60S |
| **Cycloheximide** | 80S ribosome (translocation) | Blocks eukaryotic translation (research tool) |

<image>A diagram of protein targeting via the secretory pathway. The sequence of events is shown: (1) A ribosome begins translating mRNA in the cytoplasm; the N-terminal signal peptide emerges. (2) SRP binds the signal peptide and pauses translation. (3) SRP docks with the SRP receptor on the rough ER membrane. (4) The ribosome is handed off to the translocon (Sec61 channel); translation resumes, and the polypeptide is threaded into the ER lumen. (5) Signal peptidase cleaves the signal peptide inside the lumen. (6) In the ER lumen, N-linked glycosylation occurs (oligosaccharide transferred from dolichol-P to Asn-X-Ser/Thr), disulfide bonds form (PDI), and chaperones assist folding (BiP, calnexin). (7) Properly folded proteins are packaged into COPII vesicles and transported to the Golgi for further processing (O-linked glycosylation, proteolytic processing, M6P tagging for lysosomal targeting). (8) From the Golgi, proteins are sorted to their final destinations: secretory vesicles (constitutive or regulated secretion), plasma membrane, or lysosomes (via M6P receptor). An inset shows the misfolded protein pathway: ERAD retrotranslocation, ubiquitination, and proteasomal degradation.</image>

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