Translation and the Genetic Code: Structure, Function, and Mechanisms

Chapter 13: Translation

Overview of Gene Expression

Gene expression involves the conversion of genetic information from DNA into functional products, primarily proteins. This process occurs in two main stages: transcription and translation.

• Transcription: Produces an RNA copy of a gene.

• Translation: Uses the information in mRNA to synthesize a polypeptide.

Relationship Between Genes and Proteins

Archibald Garrod and Inborn Errors of Metabolism

Archibald Garrod proposed that genes are related to protein production, based on his study of alkaptonuria.

• Alkaptonuria: Patients accumulate abnormal levels of homogentisic acid due to a missing enzyme, homogentisic acid oxidase.

• Inheritance is recessive.

• First described as an inborn error of metabolism.

Beadle and Tatum’s Experiments

Beadle and Tatum investigated the relationship among genes, enzymes, and traits using Neurospora crassa.

• Wild-type cells (prototrophs) grow on minimal media.

• Mutants (auxotrophs) require supplementation with specific biosynthetic intermediates.

• Conclusion: One gene–one enzyme hypothesis (now modified).

Modern Modifications

1. Enzymes are only one category of proteins; some genes encode other types of proteins.

2. Some proteins are composed of more than one polypeptide.

   • Polypeptide denotes structure; Protein denotes function.

3. Alternative splicing allows one gene to encode multiple polypeptides.

4. Many genes encode functional RNAs.

The Genetic Code

Nature of the Genetic Code

Translation interprets the nucleotide language of mRNA into the amino acid language of proteins.

• Genetic information is coded in mRNA as groups of three nucleotides called codons.

• Codons are recognized by the anticodon in tRNA.

Reading Frame and Codon Recognition

• The start codon sets the reading frame for all remaining codons.

• Codon is in the mRNA and is recognized by the anticodon on the tRNA.

Codon Table and Special Codons

Codon	Universal Meaning	Exception
AUA	Isoleucine	Methionine in some mitochondria
UGA	Stop	Tryptophan in mitochondria
CUA, CUC, CUG, CUU	Leucine	Threonine in yeast mitochondria
UAG, UAA	Stop	Glutamine in some ciliates
UAC	Tyrosine	Phenylalanine in some mitochondria

• AUG (methionine) = start codon; codes for methionine within protein.

• UAA, UAG, UGA = stop codons (termination/nonsense codons).

Degeneracy and Universality of the Code

• The code is degenerate: synonymous codons specify the same amino acid.

• In most cases, the third base is the degenerate (wobble) base.

• The code is nearly universal, with only a few rare exceptions.

Wobble Base Pairing

• tRNA does not always follow expected complementary rules at the 3rd position of the mRNA codon.

• This allows a single tRNA to recognize more than one codon, reducing the number of tRNAs needed.

tRNA Structure and Function

Recognition Between tRNA and mRNA

• tRNAs recognize 3-base codons in mRNA and carry the corresponding amino acid.

• The anticodon is anti-parallel to the codon.

Common Structural Features of tRNAs

• Secondary structure: cloverleaf pattern with three stem-loop structures and variable regions.

• Acceptor stem with a 3' single-stranded region for amino acid attachment.

• Tertiary structure involves additional folding.

• tRNAs often contain modified nucleotides (over 60 types).

Charging of tRNAs

• Enzymes called aminoacyl-tRNA synthetases attach amino acids to tRNAs.

• There are 20 types, one for each amino acid.

• The process involves amino acid, tRNA, and ATP.

• Highly accurate: error rate < 1/100,000.

Definition

• tRNA "charging" is the addition of the correct amino acid to a specific tRNA by aminoacyl-tRNA synthetases.

Ribosome Structure and Assembly

Polyribosomes (Polysomes)

• Transcripts with multiple ribosomes translating simultaneously.

Types of Ribosomes

• Bacterial cells: one type in cytoplasm.

• Eukaryotic cells: two types (cytoplasm and organelles such as mitochondria and chloroplasts).

Comparison of Ribosomal Subunits

Organism	Large Subunit	Small Subunit
Bacteria	50S (23S rRNA, 5S rRNA, 34 proteins)	30S (16S rRNA, 21 proteins)
Eukaryotes	60S (28S rRNA, 5.8S rRNA, 5S rRNA, 49 proteins)	40S (18S rRNA, 33 proteins)

Functional Sites of Ribosomes

• P site (Peptidyl): Holds the tRNA with the growing polypeptide chain.

• A site (Aminoacyl): Holds the tRNA carrying the next amino acid.

• E site (Exit): Where discharged tRNAs leave the ribosome.

Stages of Translation

Initiation (Prokaryotes)

• Binding of mRNA to 30S subunit is facilitated by the Shine-Dalgarno sequence (complementary to 16S rRNA).

• Initiation complex forms with mRNA, initiator tRNA, and ribosomal subunits, requiring IF1, IF2, and IF3.

• Initiator tRNA is tRNAfMet (methionine covalently modified to N-formylmethionine).

• Start codon is AUG.

Initiation (Eukaryotes)

• Initiator tRNA is tRNAMet (carries methionine).

• Start codon is AUG.

• Consensus sequence for optimal start codon recognition (Kozak's rules): G C C (A/G) C C AUG G

• Initiation factor complex binds to the 5' cap in mRNA, and the assembly scans for the start codon.

• Large subunit joins to form the 80S initiation complex.

Elongation

• Amino acids are added to the polypeptide chain one at a time.

• Peptidyl transferase (component of 50S subunit, 23S rRNA) catalyzes peptide bond formation.

• Ribosome is a ribozyme (RNA with catalytic activity).

• tRNAs move through the P, A, and E sites.

• 16S rRNA (part of 30S subunit) detects incorrect tRNA at A site and prevents elongation until mispaired tRNA is released.

• Error rate: 1 mistake per 10,000 amino acids.

Polypeptide Directionality

• First amino acid has an exposed amino group (N-terminus).

• Last amino acid has an exposed carboxyl group (C-terminus).

Termination

• Occurs when a stop codon is reached in mRNA (UAA, UAG, UGA).

• Stop codons are recognized by release factors, not tRNAs.

Protein Structure

Levels of Protein Structure

• Primary structure: Sequence of amino acids.

• Secondary structure: Regular folding patterns (alpha helix, beta sheet).

• Tertiary structure: Overall 3D shape of a single polypeptide.

• Quaternary structure: Association of multiple polypeptides.

Additional Info

• Closed loop translation in eukaryotes allows efficient ribosome recycling and prevents translation of degraded mRNA.

• Bacterial translation can begin before transcription is completed due to lack of a nucleus (coupling of transcription and translation).

Key Equations and Concepts

• Peptide bond formation: $\text{Peptide bond:} \quad \text{C} = \text{O} - \text{NH}$

• Codon-anticodon pairing: $\text{Codon (mRNA):} \quad 5'\text{UCG}3'$ $\text{Anticodon (tRNA):} \quad 3'\text{AGC}5'$

Tables

Table: Various Protein Factors Involved during Translation in E. coli

Process	Factor	Role
Initiation of translation	IF1	Binds to 30S subunit and prevents aminoacyl tRNA from binding to the A site prematurely
Initiation of translation	IF2	Binds the initiator tRNA and transfers it to the P site of the 30S-mRNA complex; releases from complex upon GTP hydrolysis
Initiation of translation	IF3	Binds to 30S subunit, preventing it from associating with the 50S subunit prematurely
Elongation of polypeptide	EF-Tu	Binds GTP; brings aminoacyl tRNA to the A site of the ribosome
Elongation of polypeptide	EF-Ts	Regulates EF-Tu activity
Elongation of polypeptide	EF-G	Stimulates translocation; GTP-dependent
Termination of translation and release of polypeptide	RF1	Catalyzes release of the polypeptide chain from tRNA and dissociation of the translation complex; specific for UAA and UAG termination
Termination of translation and release of polypeptide	RF2	Behaves like RF1; specific for UGA and UAA codons
Termination of translation and release of polypeptide	RF3	Stimulates RF1 and RF2 release