Gene Expression II: The Genetic Code and Protein Synthesis

19.1 The Genetic Code

The genetic code is the set of rules by which the nucleotide sequence of DNA is translated into the amino acid sequence of proteins. This section explores the historical experiments, properties, and structure of the genetic code.

• One Gene–One Enzyme Hypothesis: Beadle and Tatum demonstrated that genes encode enzymes by studying Neurospora crassa mutants unable to synthesize certain amino acids or vitamins.

• One Gene–One Polypeptide Hypothesis: Ingram and Pauling showed that sickle-cell anemia results from a single amino acid change in hemoglobin, refining the hypothesis to "one gene–one polypeptide." Yanofsky further confirmed this in bacteria.

• Gene Function Complexity: Eukaryotic genes often undergo alternative splicing, producing multiple polypeptides from one gene. Some genes encode functional RNAs rather than proteins.

• Triplet Code: The genetic code is based on triplets (codons) of nucleotides, allowing 64 possible combinations for 20 amino acids.

• Frameshift Mutations: Insertions or deletions (indels) shift the reading frame, causing mutant phenotypes unless three nucleotides are added or removed.

• Degeneracy and Nonoverlapping: The code is degenerate (multiple codons per amino acid) and nonoverlapping (each nucleotide is part of only one codon).

• Codon Dictionary: Of 64 codons, 61 specify amino acids, 1 (AUG) is a start codon (methionine), and 3 (UAA, UAG, UGA) are stop codons.

• Universality: The genetic code is nearly universal, with minor exceptions in mitochondria and some bacteria.

• Codon Usage Bias: Some synonymous codons are preferred, affecting translation efficiency.

Example: Sickle-cell anemia is caused by a missense mutation changing glutamic acid to valine in hemoglobin.

19.2 Translation: The Cast of Characters

Translation is the process by which the information in mRNA is used to assemble a polypeptide. Several molecular players are involved:

• Ribosomes: Complexes of rRNA and protein, composed of large and small subunits. Eukaryotic ribosomes are larger and more complex than prokaryotic ribosomes.

• tRNA: Adaptor molecules that bring amino acids to the ribosome. Each tRNA is charged with its specific amino acid by an aminoacyl-tRNA synthetase.

• Aminoacyl-tRNA Synthetases: Enzymes that attach amino acids to their corresponding tRNAs using ATP hydrolysis.

• mRNA: Encodes the sequence of amino acids; contains untranslated regions (UTRs) at both ends.

• Protein Factors: Facilitate initiation, elongation, and termination of translation.

Key Sites on Ribosome:

• A site (Aminoacyl): Binds incoming aminoacyl-tRNA

• P site (Peptidyl): Holds tRNA with growing peptide chain

• E site (Exit): Where tRNA exits after releasing its amino acid

Wobble Hypothesis: Flexibility in base pairing at the third codon position allows fewer tRNAs to recognize multiple codons. Inosine in tRNA can pair with U, C, or A.

Example: tRNA anticodon 3'-UAI-5' can recognize codons AUU, AUC, and AUA (isoleucine).

19.3 The Mechanism of Translation

Translation occurs in three stages: initiation, elongation, and termination. The process differs between prokaryotes and eukaryotes.

• Initiation: Assembly of ribosome, mRNA, and initiator tRNA. In bacteria, the Shine-Dalgarno sequence helps position the start codon. In eukaryotes, the 5' cap and Kozak sequence are important.

• Elongation: Cycles of aminoacyl-tRNA binding, peptide bond formation (catalyzed by rRNA ribozyme), and translocation. Energy is provided by GTP hydrolysis.

• Termination: Release factors recognize stop codons, triggering release of the polypeptide and ribosome disassembly.

Polyribosomes: Multiple ribosomes translate a single mRNA simultaneously, forming a polysome for efficient protein synthesis.

Energy Use: Each peptide bond formation and elongation cycle uses at least four high-energy bonds (ATP/GTP).

Example: In bacteria, initiation involves IF1, IF2 (with GTP), and IF3; in eukaryotes, eIFs and the 5' cap are critical.

19.4 Mutations and Translation

Mutations are changes in the nucleotide sequence that can affect translation and protein function. Types include:

• Missense Mutation: Changes one amino acid (e.g., sickle-cell anemia).

• Nonsense Mutation: Converts an amino acid codon to a stop codon, causing premature termination.

• Nonstop Mutation: Changes a stop codon to an amino acid codon, resulting in extended translation.

• Frameshift Mutation: Indels shift the reading frame, altering downstream amino acids.

• Silent Mutation: Alters a codon without changing the amino acid (synonymous).

• Larger-Scale Mutations: Duplications, inversions, and translocations affect larger DNA segments.

Suppressor tRNA: Mutant tRNAs can recognize stop codons and insert amino acids, suppressing nonsense mutations. Efficiency is limited to prevent cell death.

Nonsense-Mediated Decay: Eukaryotic cells degrade mRNAs with premature stop codons using exon junction complexes (EJC).

Nonstop Decay: mRNAs lacking stop codons are degraded; in bacteria, tmRNA targets the protein for destruction.

19.5 Posttranslational Processing

After translation, polypeptides often undergo modifications to become functional proteins.

• N-terminal Processing: Removal of N-formyl group (bacteria) or methionine (eukaryotes).

• Proteolytic Cleavage: Removal of blocks of amino acids (e.g., zymogen activation, insulin maturation).

• Chemical Modifications: Methylation, phosphorylation, acetylation, ubiquitination, glycosylation, addition of prosthetic groups (e.g., heme in hemoglobin).

• Protein Splicing: Removal of inteins and joining of exteins, similar to RNA splicing.

• Folding: Molecular chaperones (Hsp70, Hsp60) assist in proper folding and prevent aggregation.

Example: Insulin is processed from preproinsulin by removal of N-terminal and internal sequences, forming mature insulin with disulfide bonds.

Key Equations and Concepts

• Energy for Peptide Bond Formation:

• per phosphoanhydride bond

• Codon Number:

• possible codons (triplet code)

Additional info: The notes include inferred details about ribosome structure, translation factors, and mutation effects for completeness.