The Genetic Code, Translation, and Gene Mutations: A Comprehensive Study Guide

Isolated Congenital Asplenia and the Significance of Translation

• Overview of the Spleen: The spleen is a brownish organ weighing approximately $\frac{1}{3}\,lb$, located in the upper left abdomen. Its primary functions include storing blood, filtering out bacteria, and removing old blood cells. While adults can survive without one (often lost due to trauma), they remain at an increased risk of infection.

• Isolated Congenital Asplenia (ICA): This is a rare, autosomal dominant disorder where children are born without a spleen.

  • Affected children are highly susceptible to life-threatening bacterial infections that the immune system would normally eliminate.

  • These infections often develop into systemic raging infections, frequently resulting in death during childhood despite antibiotic treatment.

• Genetic Cause of ICA: In 2013, researchers used DNA sequencing to compare the coding DNA of 23 individuals with ICA against 508 normal individuals.

  • The disorder was associated with mutations in the gene encoding Ribosomal Protein SA (RPSA).

  • RPSA is one of the 33 proteins constituting the small subunit of the ribosome, which is the molecular machine responsible for protein synthesis (translation).

  • Ribosomopathies: Diseases like ICA that are caused by defective ribosomes.

  • Observed Mutations in RPSA:

    • Premature stop codons (halting translation).

    • Frameshift mutations (altering the mRNA reading frame).

    • Amino acid substitutions (altering the protein sequence).

• Scientific Paradox: While RPSA mutations exist in every cell and translation is a universal necessity, ICA specifically affects only the development of the spleen. The reason for this organ-specific impact remains an open area of research.

15.1 The Evolution of the Relation Between Genes and Proteins

• Archibald Garrod (1908): The first to propose that genes encode enzymes, though his theory was largely ignored by his contemporaries.

• Beadle and Tatum (1940s): Conducted definitive research using the bread mold Neurospora crassa to establish the one gene, one enzyme hypothesis.

  • Advantages of Neurospora: It is easy to cultivate and is haploid for much of its life cycle, allowing even recessive mutations to be observed immediately in the phenotype (no masking by a dominant allele).

• Auxotrophs: Mutants that are nutritionally deficient and cannot grow on a minimal medium (inorganic salts, sucrose, and biotin) because they cannot synthesize essential biological molecules.

• Experimental Procedure (Beadle and Tatum):

  1. Irradiated Neurospora spores with X-rays to induce mutations.

  2. Transferred spores to complete medium (providing all nutrients) to ensure growth.

  3. Transferred spores to minimal medium; those that failed to grow were identified as auxotrophic mutants.

  4. To identify the specific defect, mutants were placed in tubes of minimal medium supplemented with a single specific amino acid. Growth indicated the fungus lacked the ability to synthesize that specific supplement.

• The Arginine Synthesis Pathway (Srb and Horowitz): Used genetic dissection to map the multistep pathway for arginine synthesis.

  • Groups of Mutants:

    • Group I: Grew on minimal medium + ornithine, citrulline, or arginine. Mutation blocks the earliest step (precursor $\rightarrow$ ornithine).

    • Group II: Grew on citrulline or arginine, but not ornithine. Mutation blocks the step (ornithine $\rightarrow$ citrulline).

    • Group III: Grew only on arginine. Mutation blocks the final step (citrulline $\rightarrow$ arginine).

  • Conclusion: Each gene encodes a separate enzyme. Because some proteins are non-enzymatic or consist of multiple subunits, this was refined to the one gene, one polypeptide hypothesis.

Structure and Function of Proteins

• General Roles: Proteins act as biological catalysts (enzymes like luciferase), structural supports (fibroin in spiderwebs), toxins (ricin in castor beans), and transporters.

• Amino Acid Structure: All 20 common amino acids consist of a central carbon atom ($C_{\alpha}$) bonded to:

  1. An amino group ($NH_3^{+}$).

  2. A carboxyl group ($COO^{-}$).

  3. A hydrogen atom ($H$).

  4. A variable radical (R) group that determines chemical properties.

• Peptide Bonds: Covalent bonds that link the carboxyl group of one amino acid to the amino group of the next.

• Levels of Organization:

  • Primary Structure: The specific linear sequence of amino acids.

  • Secondary Structure: Local folding (alpha helix or beta pleated sheet) driven by hydrogen bonding.

  • Tertiary Structure: The overall three-dimensional shape of a single polypeptide.

  • Quaternary Structure: The association of two or more polypeptide chains (e.g., hemoglobin).

• Protein Domains: Discrete functional units within a protein formed by specific groups of amino acids.

15.2 The Nature of the Genetic Code

• The Triplet Code: A codon consists of three nucleotides.

  • If a codon were 1 base, only 4 codons would exist ($4^1$).

  • If 2 bases, only 16 ($4^2$).

  • 3 bases provide 64 possible codons ($4^3$), sufficient for 20 amino acids.

• Breaking the Code (Nirenberg and Matthaei): Used polynucleotide phosphorylase to create synthetic homopolymers.

  • Poly(U): Resulted in a polypeptide chain of ONLY phenylalanine ($UUU = Phe$).

  • Poly(C): Resulted in proline ($CCC = Pro$).

  • Poly(A): Resulted in lysine ($AAA = Lys$).

• Random Copolymers: Mixing nucleotides in set ratios (e.g., $4:1$ C to A) helped determine base composition but not order.

  • Probability of a specific codon (e.g., 2 Cs and 1 A): $\frac{4}{5} \times \frac{4}{5} \times \frac{1}{5} = \frac{16}{125} \approx 0.13$

• Nirenberg and Leder (1964): Used ribosome-bound tRNAs and short synthetic mRNAs (triplets).

  • The triplet would bind a ribosome and attract the specific tRNA with the complementary anticodon.

  • The mixture was passed through a nitrocellulose filter; only the large ribosome complexes (with bound tRNA) stuck to the filter.

• Features of the Code:

  • Degeneracy: The code is redundant; multiple codons can specify one amino acid (e.g., leucine has 6 codons).

  • Sense Codons: The 61 codons that specify amino acids.

  • Termination (Stop) Codons: UAA, UAG, and UGA; these signal the end of translation.

  • Initiation (Start) Codon: Usually AUG. In bacteria, it encodes N-formylmethionine ($fMet$); in eukaryotes, it encodes unformylated methionine ($Met$).

  • Wobble Hypothesis (Crick, 1966): Flexibility in base pairing at the third position of the codon. For example, a $G$ in the 5' position of the tRNA anticodon can pair with either $C$ or $U$ in the 3' position of the mRNA codon.

  • Reading Frame: The way the sequence is partitioned into triplets. It is set by the initiation codon.

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

15.3 The Process of Translation

• Directionality: Translation proceeds from the 5' to 3' end of mRNA. Proteins are synthesized from the amino (N) to carboxyl (C) end.

• Stage 1: tRNA Charging:

  • Aminoacyl-tRNA synthetases (20 specific enzymes) link amino acids to their corresponding tRNAs.

  • Requires energy from ATP: $\text{amino acid} + \text{tRNA} + \text{ATP} \rightarrow \text{aminoacyl-tRNA} + \text{AMP} + \text{PP}_i$.

  • The amino acid attaches to the adenine nucleotide at the 3' end (CCA) of the tRNA.

• Stage 2: Initiation:

  • Bacteria: The small 30S ribosomal subunit binds to the Shine-Dalgarno sequence in the mRNA 5' UTR. Initiation factors (IF-1, IF-2-GTP, IF-3) assist. The initiator tRNA ($fMet-tRNA_i^{Met}$) binds AUG. Finally, the 50S subunit joins to form the 70S initiation complex.

  • Eukaryotes: The small subunit binds the 5' cap (aided by the Cap-Binding Complex and eIF-4E) and scans the mRNA for the first AUG within the Kozak sequence ($5'\text{-ACCAUGG-3'}$). The poly(A) tail interacts with the cap to form a "closed loop" structure.

• Stage 3: Elongation:

  • Sites: Aminoacyl (A), Peptidyl (P), and Exit (E).

  • Step 1: Charged tRNA enters the A site (aided by EF-Tu and GTP).

  • Step 2: Peptide bond formation catalyzed by the 23S rRNA (bacteria) or 28S rRNA (eukaryotes) acting as a ribozyme.

  • Step 3: Translocation (aided by EF-G and GTP hydrolysis). The ribosome moves one codon downstream; tRNA moves from P to E, and A to P.

• Stage 4: Termination:

  • Occurs when the A site reaches a stop codon.

  • Release Factors (RFs) bind instead of tRNA. In E. coli, RF-1 recognizes UAA/UAG, and RF-2 recognizes UAA/UGA. RF-3-GTP facilitates the release of the factors and dissociation of the ribosome.

Chapter 18: Gene Mutations and Lou Gehrig's Disease

• Amyotrophic Lateral Sclerosis (ALS): A progressive disease causing muscle wasting due to motor neuron degeneration.

• C9orf72 Gene: Mutations of this gene on chromosome 9 are a major cause of familial ALS and frontotemporal dementia (FTD).

• Expanding Nucleotide Repeats: The mutation involves an increase in copies of the sequence GGGGCC.

  • Normal: $2\text{ to }23$ repeats.

  • ALS Patients: $700\text{ to }1600$ repeats.

• Toxic Translation: The GGGGCC repeats are transcribed and translated without a start codon in all three reading frames on both strands. This generates five types of repeating dipeptide proteins: glycine-alanine, glycine-proline, proline-alanine, glycine-arginine, and proline-arginine. The dipeptides with arginine are believed to be toxic to nerve cells.

Categories and Types of Mutations

• Classification by Tissue Type:

  • Somatic Mutations: Occur in non-reproductive cells. Passed to daughter cells via mitosis, creating a clone of mutant cells. Can lead to cancer.

  • Germ-line Mutations: Occur in cells that produce gametes. Inherited by offspring and present in every cell of the offspring's body.

• Molecular Types of Mutations:

  • Base Substitutions:

    • Transition: Purine to Purine ($A \leftrightarrow G$) or Pyrimidine to Pyrimidine ($C \leftrightarrow T$).

    • Transversion: Purine to Pyrimidine (e.g., $A \rightarrow C$) or Pyrimidine to Purine.

  • Insertions and Deletions (Indels): Additions or removals of nucleotides.

    • Frameshift mutation: Changes the reading frame (happens if the indel is not a multiple of 3).

    • In-frame indel: Deletion/insertion of a multiple of 3; reading frame remains intact.

• Functional Effects:

  • Missense: Changes one amino acid to another.

  • Nonsense: Changes a sense codon into a stop codon (truncates the protein).

  • Silent: Changes a codon to a synonymous one; amino acid sequence is unchanged.

  • Neutral: Missense mutation that doesn't affect protein function.

  • Loss-of-function: Causes complete or partial absence of normal function (usually recessive).

  • Gain-of-function: Produces a new trait or function (usually dominant).

Suppressor Mutations

• Definitions: A suppressor mutation hides the effect of a primary mutation at a different site.

• Intragenic Suppressor: Occurs in the same gene as the original mutation (e.g., a one-base insertion suppressing a one-base deletion).

• Intergenic Suppressor: Occurs in a different gene.

  • Example: A mutation in a tRNA gene allows its anticodon to recognize a stop codon produced by a nonsense mutation in another gene, allowing translation to continue.

Mutation Rates and Causes

• Mutation Rate: Expressed as mutations per biological unit (e.g., $4/100,000$ gametes for achondroplasia).

• Factors: Frequency of DNA changes, efficacy of DNA repair, and detectability of the phenotype.

• Spontaneous Replication Errors:

  • Tautomeric Shifts: Protons shift positions in bases, allowing anomalous pairings (e.g., C pairing with A).

  • Wobble: Non-standard pairings due to structural flexibility.

  • Strand Slippage: Loop formation during replication leading to indels.

  • Unequal Crossing Over: Misalignment during meiosis resulting in an insertion on one chromosome and a deletion on the other.

• Spontaneous Chemical Changes:

  • Depurination: Loss of a purine base; often results in an adenine being incorrectly inserted opposite the empty site durante la replicaci3n.

  • Deamination: Loss of an amino group. Deamination of cytosine produces uracil ($C \rightarrow U$), leading to transitions. Deamination of 5-methylcytosine produces thymine ($5mC \rightarrow T$).

Chemically Induced Mutations and Radiation

• Mutagens:

  • Base Analogs: Chemicals similar to bases. 5-bromouracil (5BU) is a thymine analog that can mispair with guanine.

  • Alkylating Agents: Add alkyl groups (e.g., Ethylmethanesulfonate [EMS]).

  • Deaminating Chemicals: Nitrous acid ($HNO_2$).

  • Hydroxylamine: Specifically hydroxyla cytosines, leading to $C \cdot G \rightarrow T \cdot A$ transitions.

  • Intercalating Agents: Proflavin, acridine orange, and ethidium bromide wedge between bases, causing single-base indels/frameshifts.

• Radiation:

  • Ionizing Radiation (X-rays): Dislodges electrons, creates free radicals, and breaks phosphodiester bonds.

  • Ultraviolet (UV) Light: Causes pyrimidine dimers (mostly thymine dimers) that distort DNA and block replication.

  • SOS System: A high-error bypass mechanism in bacteria that allow replication to proceed past dimers at the cost of accuracy.

The Ames Test

• Purpose: To screen chemicals for carcinogenic potential by testing their mutagenicity in bacteria.

• Method: Uses auxotrophic strains of Salmonella typhimurium ($his^{-}$) that cannot synthesize histidine.

• Logic: If a chemical is a mutagen, it will cause a reverse mutation ($his^{-} \rightarrow his^{+}$), allowing the bacteria to grow on histidine-lacking medium. Mammalian liver enzymes are added to simulate human metabolism.

Transposable Elements (Transposons)

• General Features: Mobile DNA sequences. They generate flanking direct repeats ($3\text{ to }12\,bp$) in the target DNA at the point of insertion and often have terminal inverted repeats.

• Mechanisms of Transposition:

  • DNA Transposons (Class II): Move directly as DNA.

    • Replicative: "Copy and paste" (total number of copies increases).

    • Nonreplicative: "Cut and paste" (element is excised and moved).

  • Retrotransposons (Class I): Move via an RNA intermediate using reverse transcriptase.

• Bacterial Transposons:

  • Insertion Sequences (IS): Simplest; only encode transposase.

  • Composite Transposons (Tn): A DNA segment flanked by two IS elements (e.g., Tn10 which carries tetracycline resistance).

  • Noncomposite Transposons: Lack IS elements but have inverted repeats and carry other genes (e.g., Tn3).

• Eukaryotic Transposons:

  • Ac/Ds in Corn (McClintock): Ac is autonomous (encodes transposase); Ds is nonautonomous (has deletions and requires Ac to move). Kernels become variegated as Ds transposes out of pigment-regulating genes during development.

  • Mutagenic Impact: Transposons can disrupt gene function (e.g., hemophilia caused by L1 insertion into Factor VIII) or cause chromosomal rearrangements like deletions or inversions via homologous recombination between multiple copies.