D1.2 Protein Synthesis

D1.2 Protein Synthesis

Overview

Protein synthesis is the process by which cells use genetic information in DNA to build proteins. This occurs in two main stages:

1. Transcription — DNA is copied into messenger RNA (mRNA) in the nucleus

2. Translation — mRNA is decoded by ribosomes to assemble amino acids into polypeptides

This process is often called gene expression — converting the information in a gene into a functional product.

The Central Dogma of Molecular Biology

The central dogma, proposed by Francis Crick, describes the flow of genetic information:

    DNA  →  RNA  →  Protein
         ↑      ↑
   Transcription  Translation

Key points:

• Information flows from nucleic acids to proteins

• DNA stores genetic information

• RNA acts as an intermediary messenger

• Proteins carry out cellular functions

• Information does NOT flow from protein back to nucleic acid (with rare exceptions like reverse transcriptase in retroviruses)

Genes and the Genetic Code

What is a Gene?

A gene is a sequence of DNA nucleotides that codes for a polypeptide (or functional RNA).

Gene structure (simplified):

• Promoter — Regulatory sequence where transcription begins

• Coding sequence — Contains information for polypeptide

• Terminator — Signals end of transcription

• Introns — Non-coding sequences (eukaryotes only)

• Exons — Coding sequences

The Genetic Code

The genetic code is the set of rules by which information in DNA/mRNA is translated into amino acid sequences.

Codons

• A codon is a sequence of three nucleotides (triplet)

• Each codon specifies one amino acid (or stop signal)

• mRNA is read in codons during translation

Properties of the Genetic Code

The Codon Table

Start codon: AUG (codes for methionine; initiates translation)

Stop codons: UAA, UAG, UGA (terminate translation; do not code for amino acids)

Degeneracy pattern:

• Third position often "wobbles" — can vary without changing amino acid

• First and second positions more critical for specificity

• Provides buffer against point mutations

Types of RNA

Three main types of RNA are involved in protein synthesis:

mRNA (Messenger RNA)

Structure:

• Single-stranded polynucleotide

• Complementary to template DNA strand

• Same sequence as coding strand (except U instead of T)

• Contains codons read during translation

Features (eukaryotic mRNA):

• 5' cap — Modified guanine nucleotide; protects from degradation; aids ribosome binding

• 5' UTR — Untranslated region before start codon

• Coding sequence — Start codon to stop codon

• 3' UTR — Untranslated region after stop codon

• Poly-A tail — String of adenine nucleotides; protects from degradation; aids export from nucleus

tRNA (Transfer RNA)

Structure:

• Single strand that folds into characteristic cloverleaf shape (2D) or L-shape (3D)

• ~75–95 nucleotides long

• Contains modified bases

• Held by intramolecular base pairing (hydrogen bonds)

Key features:

Aminoacyl-tRNA synthetase:

• Enzyme that attaches correct amino acid to tRNA

• One specific synthetase for each amino acid

• Recognises both amino acid AND its corresponding tRNA(s)

• Uses ATP energy

• Critical for translation accuracy

Charged tRNA = tRNA with amino acid attached

rRNA (Ribosomal RNA)

Function:

• Structural component of ribosomes

• Catalytic activity (ribozyme) — forms peptide bonds

• Most abundant RNA in cells (~80% of total RNA)

Types:

• Prokaryotes: 16S, 23S, 5S rRNA

• Eukaryotes: 18S, 28S, 5.8S, 5S rRNA

• Numbers refer to sedimentation rates (Svedberg units)

Ribosomes

Ribosomes are the molecular machines that carry out translation.

Structure

Note: Svedberg units (S) are not additive because they measure sedimentation rate, not mass.

Functional Sites

Polyribosomes (Polysomes)

• Multiple ribosomes can translate the same mRNA simultaneously

• Increases protein production efficiency

• Ribosomes spaced ~80 nucleotides apart

• Form a "string of beads" appearance on mRNA

Transcription

Transcription is the synthesis of RNA using DNA as a template. It occurs in the nucleus (eukaryotes) or cytoplasm (prokaryotes).

Overview

$\text{DNA (template strand)} \xrightarrow{\text{RNA polymerase}} \text{mRNA}$

Key enzyme: RNA polymerase

DNA Strands Terminology

RNA polymerase reads the template strand 3'→5' and synthesises RNA 5'→3'.

Stages of Transcription

1. Initiation

Promoter recognition:

• RNA polymerase binds to promoter sequence upstream of gene

• Prokaryotes: Sigma factor helps recognise promoter (e.g., TATA box at −10 and −35)

• Eukaryotes: Transcription factors bind first; help position RNA polymerase

DNA unwinding:

• Local unwinding of DNA double helix (~17 bp)

• Creates transcription bubble

• Template strand exposed

First nucleotides:

• RNA polymerase begins adding ribonucleotides

• No primer needed (unlike DNA polymerase)

• Starts at +1 position (transcription start site)

2. Elongation

RNA synthesis:

1. RNA polymerase moves along template strand 3'→5'

2. Reads template bases

3. Adds complementary ribonucleotides to 3' end of growing RNA

4. RNA synthesised 5'→3'

5. Uses ribonucleoside triphosphates (ATP, GTP, CTP, UTP)

6. Two phosphates released per nucleotide (provides energy)

Base pairing rules:

Transcription bubble:

• DNA unwinds ahead of polymerase

• DNA rewinds behind polymerase

• Only ~8–9 bp of DNA-RNA hybrid at any time

• RNA peels off as it's synthesised

Speed: ~40–80 nucleotides per second (slower than replication)

3. Termination

Prokaryotes:

• Rho-independent: Hairpin loop forms in RNA; destabilises polymerase binding

• Rho-dependent: Rho protein catches up to paused polymerase; releases RNA

Eukaryotes:

• RNA polymerase transcribes past poly-A signal (AAUAAA)

• RNA is cleaved at specific site

• Poly-A tail added by poly-A polymerase

• RNA polymerase eventually falls off

Comparing Transcription and Replication

Post-Transcriptional Modification (Eukaryotes)

In eukaryotes, the primary transcript (pre-mRNA) undergoes several modifications before leaving the nucleus:

1. 5' Capping

Process:

• Modified guanine nucleotide (7-methylguanosine) added to 5' end

• Added in reverse orientation (5'-5' linkage)

• Occurs during transcription (co-transcriptional)

Functions:

• Protects mRNA from degradation by exonucleases

• Recognised by ribosome for translation initiation

• Aids export from nucleus

2. 3' Polyadenylation

Process:

• Poly-A signal (AAUAAA) recognised

• mRNA cleaved ~10–30 nucleotides downstream

• Poly-A polymerase adds 100–250 adenine nucleotides (poly-A tail)

Functions:

• Protects 3' end from degradation

• Aids export from nucleus

• Influences mRNA stability and translation efficiency

• Tail shortens over time → signals degradation

3. Splicing

The problem: Eukaryotic genes contain introns (non-coding sequences) interspersed with exons (coding sequences).

Introns must be removed and exons must be joined before translation.

The Spliceosome:

• Large complex of proteins and small nuclear RNAs (snRNAs)

• snRNAs + proteins = snRNPs (small nuclear ribonucleoproteins, "snurps")

• Recognises splice sites at intron-exon boundaries

• Catalyses splicing reaction

Splicing mechanism:

1. Spliceosome assembles at 5' splice site

2. Intron forms a lariat (loop) structure

3. 5' end of intron cut and joined to branch point (A) within intron

4. 3' end of intron cut

5. Exons joined together

6. Intron released and degraded

7. Spliceosome disassembles

Pre-mRNA:  Exon 1 ──── Intron ──── Exon 2

                         ↓ Splicing

Mature mRNA:  Exon 1 ═══════ Exon 2
                   (joined together)

Alternative Splicing

• Different combinations of exons can be included in mature mRNA

• One gene can produce multiple different proteins

• Greatly increases proteome diversity

• ~95% of human genes undergo alternative splicing

Example: The human DSCAM gene can produce >38,000 different proteins through alternative splicing

Types of alternative splicing:

• Exon skipping (most common)

• Alternative 5' or 3' splice sites

• Intron retention

• Mutually exclusive exons

Summary: Pre-mRNA to Mature mRNA

Pre-mRNA (primary transcript):
5'───[Exon1]──[Intron]──[Exon2]──[Intron]──[Exon3]───3'

                    ↓ Processing

Mature mRNA:
5' Cap ─── 5'UTR ─── [Exon1][Exon2][Exon3] ─── 3'UTR ─── AAAAAAA (Poly-A tail)

Translation

Translation is the synthesis of a polypeptide using the information in mRNA. It occurs on ribosomes in the cytoplasm (or on rough ER for secreted proteins).

Overview

$\text{mRNA} + \text{tRNAs} + \text{Amino acids} \xrightarrow{\text{Ribosome}} \text{Polypeptide}$

Requirements for Translation

Stages of Translation

1. Initiation

Assembly of translation machinery:

1. Small ribosomal subunit binds to mRNA

   • Prokaryotes: Binds to Shine-Dalgarno sequence upstream of start codon

   • Eukaryotes: Recognises 5' cap; scans for start codon (AUG)

2. Initiator tRNA (carrying methionine) binds to start codon (AUG)

   • Anticodon UAC pairs with codon AUG

   • Positioned in P site

3. Large ribosomal subunit joins

   • Forms complete ribosome

   • Initiator tRNA in P site

   • A site empty and ready

Initiation factors (IFs in prokaryotes; eIFs in eukaryotes) facilitate assembly

Start codon: AUG

• Codes for methionine (Met)

• In prokaryotes: formyl-methionine (fMet)

• Sets the reading frame for translation

2. Elongation

Cyclic process that adds amino acids one at a time:

Step 1: Codon recognition

• Charged tRNA enters A site

• Anticodon must be complementary to mRNA codon

• Correct base pairing triggers GTP hydrolysis

• tRNA accepted into A site

Step 2: Peptide bond formation

• Catalysed by peptidyl transferase (rRNA in large subunit — ribozyme)

• Amino acid in P site transferred to amino acid in A site

• Peptide bond forms between carboxyl group (P site) and amino group (A site)

• Growing polypeptide now attached to tRNA in A site

Step 3: Translocation

• Ribosome moves one codon (3 nucleotides) along mRNA in 5'→3' direction

• Requires elongation factors and GTP hydrolysis

• tRNA in A site moves to P site (carrying polypeptide)

• tRNA in P site moves to E site (now uncharged)

• tRNA exits from E site

• New codon exposed in empty A site

• Cycle repeats

Step 1:          Step 2:           Step 3:
  E   P   A        E   P   A         E   P   A
  │   │   │        │   │   │         │   │   │
  ○  Met  ○        ○  Met-Ala        ○   ○  Met-Ala
      │   │            │   │             │   │
     tRNA tRNA       tRNA tRNA          tRNA tRNA
      ↓   ↓            ↓   ↓             ↓   ↓
─────AUG─GCU─────  ─────AUG─GCU─────  ─────AUG─GCU─────

Elongation rate: ~15–20 amino acids per second (prokaryotes); slower in eukaryotes

3. Termination

Stop codon recognition:

1. One of three stop codons (UAA, UAG, UGA) enters A site

2. No tRNA has complementary anticodon for stop codons

3. Release factor (protein) binds to stop codon in A site

4. Release factor has shape similar to tRNA

Polypeptide release:

5. Release factor triggers hydrolysis of bond between polypeptide and tRNA in P site

6. Polypeptide released from ribosome

7. Ribosomal subunits dissociate

8. mRNA released

9. Components can be recycled

Summary of Translation

Post-Translational Modification

The polypeptide chain released from the ribosome often requires further processing to become a functional protein:

Types of Modifications

Protein Targeting

Proteins are directed to their correct location:

Signal peptide:

• Short N-terminal sequence (~15–30 amino acids)

• Recognised by signal recognition particle (SRP)

• Directs ribosome to rough ER

• Cleaved off after translation

Comparing Transcription and Translation

Prokaryotic vs Eukaryotic Protein Synthesis

Coupled transcription-translation in prokaryotes:

• No nuclear membrane separating processes

• Ribosomes bind to mRNA while it's still being transcribed

• Multiple ribosomes can translate same mRNA simultaneously

• Very efficient; rapid response to environmental changes

One Gene–One Polypeptide Hypothesis

Historical Development

Beadle and Tatum (1940s):

• Studied Neurospora crassa (bread mould)

• Created mutants using X-rays

• Found mutants that couldn't synthesise specific amino acids

• Each mutant lacked one enzyme

• Proposed: One gene = one enzyme

Refinements:

• Not all proteins are enzymes → "one gene–one protein"

• Some proteins have multiple polypeptides → "one gene–one polypeptide"

• Some genes code for functional RNAs (not proteins) → further exceptions

• Alternative splicing → one gene can code for multiple polypeptides

Modern understanding:

• Genes code for polypeptides OR functional RNAs

• Relationship more complex than originally thought

• But fundamental principle remains: genes determine protein structure

Practical Investigations

Modelling Transcription and Translation

Paper models:

• Create DNA template strand

• Match mRNA nucleotides to template

• Use codon table to determine amino acids

• Build polypeptide sequence

Analysing Sequences

Given DNA sequence, determine:

1. Template strand vs coding strand

2. mRNA sequence (complement of template, U for T)

3. tRNA anticodons (complement of mRNA codons)

4. Amino acid sequence (using codon table)

Example:

DNA coding strand: 5'—ATG—GCA—TTA—TAA—3' DNA template strand: 3'—TAC—CGT—AAT—ATT—5' mRNA: 5'—AUG—GCA—UUA—UAA—3' Amino acids: Met—Ala—Leu—STOP

Examining Effects of Mutations

• Change one nucleotide in DNA sequence

• Determine effect on mRNA and amino acid sequence

• Classify mutation type (silent, missense, nonsense)

• Predict effect on protein function

Common Exam Questions

Typical Question Types

1. Describe the process of transcription (6 marks)

   • RNA polymerase binds to promoter

   • DNA unwinds; strands separate

   • RNA polymerase reads template strand 3'→5'

   • Adds complementary ribonucleotides 5'→3'

   • A pairs with U; T pairs with A; G pairs with C; C pairs with G

   • Continues until terminator sequence

   • mRNA released

2. Describe the process of translation (6 marks)

   • mRNA binds to ribosome

   • Start codon (AUG) positions initiator tRNA in P site

   • Charged tRNA enters A site; anticodon matches codon

   • Peptide bond forms between amino acids

   • Ribosome translocates along mRNA

   • Process repeats until stop codon reached

   • Polypeptide released

3. Explain the roles of mRNA, tRNA, and rRNA (4 marks)

   • mRNA: carries genetic code from DNA to ribosome; contains codons

   • tRNA: brings amino acids to ribosome; has anticodon that matches codon

   • rRNA: structural component of ribosome; catalyses peptide bond formation

4. Describe post-transcriptional modification in eukaryotes (4 marks)

   • 5' capping: modified guanine added; protects mRNA; aids ribosome binding

   • Polyadenylation: poly-A tail added to 3' end; protects from degradation

   • Splicing: introns removed; exons joined; by spliceosome

5. Explain the properties of the genetic code (4 marks)

   • Triplet: three nucleotides code for one amino acid

   • Degenerate: multiple codons for same amino acid

   • Universal: same in almost all organisms

   • Non-overlapping: each nucleotide in only one codon

6. Compare transcription and translation (4 marks)

   • Transcription: DNA→RNA; in nucleus; RNA polymerase; produces mRNA

   • Translation: mRNA→polypeptide; in cytoplasm; ribosomes; produces protein

   • Both involve complementary base pairing

   • Both synthesise in 5'→3' / N→C direction

Key Terminology Glossary

Summary Flowchart

                    NUCLEUS
                       │
        DNA (gene) ────┼──→ Transcription
                       │         │
                       │         ↓
                       │    Pre-mRNA
                       │         │
                       │    Processing:
                       │    • 5' capping
                       │    • Splicing
                       │    • Polyadenylation
                       │         │
                       │         ↓
                       │    Mature mRNA
                       │         │
        ───────────────┼─────────┼───────────────
                       │         │
                   CYTOPLASM     │
                                 ↓
                           Translation
                                 │
                    ┌────────────┼────────────┐
                    ↓            ↓            ↓
                Ribosome      tRNAs       Amino acids
                    │            │            │
                    └────────────┼────────────┘
                                 │
                                 ↓
                           Polypeptide
                                 │
                    Post-translational modification
                                 │
                                 ↓
                        Functional Protein