Genetic Code and tRNA Function

• The genetic code underlies transferring DNA base sequences to amino acid sequences in polypeptides with translation occurring at the ribosome, directing amino acid polymerization.

Genetic Code Features

• Universality: The code is generally the same across organisms, with minor exceptions in plants, microorganisms, and mitochondria.
• Nonoverlapping and commaless: It is read continuously, three bases (a triplet codon) at a time, without spacers.
• Specificity: Each codon corresponds to a single amino acid.
• Redundancy (degeneracy): Several codons can represent the same amino acid.

Transfer RNA (tRNA) Adaptor Function

• There are 31 tRNA species that differ in anticodon sequences to match mRNA codons and in recognition by aminoacyl-tRNA synthetases.
• All tRNAs share common structural features, including the acceptor arm, anticodon loop, D arm, and TψC arm
• Acceptor arm: The CCA 3'-hydroxyl terminus is the site for amino acid attachment, added posttranscriptionally.
• Anticodon loop: It contains a triplet of bases that pair with the mRNA codon in an antiparallel orientation.
• D arm: It is involved in recognition by aminoacyl-tRNA synthetase, named for its dihydrouracil content.
• TψC arm: It is involved in functional binding to the ribosome, named for thymine and pseudouracil bases.

Aminoacyl-tRNA Synthetases

• These enzymes covalently link the correct amino acid to the correct tRNA, a process called tRNA charging, so amino acids are activated and tRNA acylated
• Amino acid activation: ATP produces aminoacyl-adenosine monophosphate.
• Acylation: Transferring the amino acid from the aminoacyl-adenosine monophosphate.
• Aminoacyl-tRNA synthetase recognizes genetic code and amino acid simultaneously
• Amino acid attachment is based on anticodon information, following the "adaptor hypothesis."
• Cysteinyl-tRNA was charged with cysteine and then modified to alanine, sequence analysis showed the alanine was incorporated at the cysteine positions

Aminoacyl-tRNA Synthetase Proofreading

• Synthetase mistakes have the same effect as point mutations.
• Synthetases proofread and hydrolyze incorrect amino acids, as they contain hydrolytic sites that remove incorrect matches.

Wobble in Anticodon Base-Pairing

• Since there are only 31 tRNA species for 61 codons, most tRNAs recognize multiple codons due to wobble
• Wobble is nonstandard base pairing at the first (5') anticodon position and third (3') mRNA codon position, so the bases adopt alternate hydrogen bonding
• U in the anticodon can base pair with A or G on the mRNA.
• G in the anticodon can base pair with C or U on the mRNA.
• I (inosine) in the anticodon can base pair with U, C, or A on the mRNA

Mutations

• Although proofreading and repair prevent mutation, lesions remain and become heritable changes after DNA synthesis.
• Continuously proliferating cells are more prone to mutations.
• The three major categories of mutation are base change, frameshift mutation, and recombination mutation.
• Base change (substitution) mutation: Change from one base to another.
• Frameshift mutation: Alteration of codon reading frame by base addition or deletion.
• Recombination mutation: Exchange between two DNA molecules.

Base Change Mutations

• Base change (point) mutations are produced by chemical modifications to existing bases or incorporation of base analogs.
• Transition mutation: Substitution of one base type with the same type.
• Transversion mutation: Substitution of one base type with its opposite type.
• Silent mutation: No change in the protein.
• Missense mutation: Change to a similar amino acid.
• Nonsense mutation: Termination codon appears and the polypeptide is truncated.

Frameshift Mutations

• Frameshift mutations are produced by molecules that can insert (intercalate) between the normal bases to create mistakes during DNA synthesis.
• After insertion or deletion, mRNA is read out of frame, yielding a nonsense protein that can produce a termination codon.

Recombination Mutations

• Recombination is a normal process where chromosomes exchange gene alleles.
• When it occurs during meiosis, it is referred to as crossing over.
• If misalignment occurs, unequal distribution of DNA results, thus creating deletion on one strand and duplication on the other.
• An example of such an unequal crossover is the Lepore thalassemia variant allele
• A hybrid δ-β globin protein is produced by the slower δ-globin promoter and classifies the mutation as a thalassemia.

Histology - Cell Division

• Continuously dividing cells are either differentiating mitotic cells or vegetative intermitotic cells (stem cells).
• Examples of stem cells are epidermal basal cells, regenerative cells in the intestines, and bone marrow stem cells.
• Examples of differentiating mitotic cells include prickle cells in the stratum spinosum and fibroblasts during wound healing.

Protein Synthesis

• The process of peptide assembly on a ribosome using mRNA blueprint from tRNA parts
• Polypeptides undergo posttranslational modification.

Ribosome

• A ribonucleoprotein particle with similar composition in prokaryotes and eukaryotes, where ribosomal proteins and RNA will self-assemble into subunits when mixed
• Protein translation factors coordinate polypeptide initiation, elongation, and termination, so a complete ribosome contains three tRNA-binding sites.
• A (aminoacyl) site: It binds the new aminoacyl-tRNA.
• P (peptidyl) site: Binds the growing peptide attached to the most recent tRNA.
• E (exit) site: It contains deacylated tRNA, so the peptide is attached to the ribosome by tRNA during elongation.

Polypeptide Chain Elongation

• A 3-step cyclic process where new amino acids are incorporated into the growing polypeptide chain in the amino to carboxyl terminal direction.
• Aminoacyl-tRNA binding: Aminoacyl-tRNA binds with elongation factor (EF)-Tu and GTP, so ejection of deacylated tRNA from the E site transpires.
• Peptide bond formation: New aminoacyl-tRNA binds to the A site and is aligned with peptidyl-tRNA at the P site, so peptidyltransferase creates a peptide bond.
• Peptidyl tRNA translocation: Peptidyl-tRNA moves to the P site by EF-G, requiring one GTP, so mRNA and peptidyl-tRNA move one codon and the deacylated tRNA moves to the E site.
• Incorporating one aminoacyl-tRNA into a protein costs 2 GTP, so total cost for one amino acid is four high-energy bonds.
• EF-Tu-GTP complex becomes EF-Tu-guanosine diphosphate, regenerated by EF-Ts
• Some toxins and antibiotics disrupt protein synthesis

Polypeptide Chain Initiation (Prokaryotic)

• The process aligns the first amino acid with the initiation codon and subunit association.
• The first amino acid is located in the P site, where the process occurs in two stages of 30S and 70S initiation complexes
• 30S initiation complex: N-formylmethionine (fMet), mRNA, GTP, and initiation factors (IF)-1 and -3, so the P site now contains fMet-tRNA aligned with the AUG codon and correct alignment is based on the Shine-Dalgarno sequence.
• 70S initiation complex: As the 50S subunit binds and GTP is hydrolyzed, the complete ribosome contains fMet tRNA in the P site and A site is ready to receive the next aminoacyl-tRNA
• tRNAf carries fMet, produced from methionyl-tRNA by transformylase
• Tu does not form a binding complex with fMet-tRNA, so Met insertion at interior sites uses unmodified methionyl-tRNA bound by Tu.

Polypeptide Chain Initiation (Eukaryotic)

• A similar initiation process that uses more proteins
• Cap-binding proteins recognize the methylguanosine cap on the mRNA
• A specialized initiator tRNA specific for methionine is used but is not formylated.
• eIF-3 helps align the AUG codon with methionyl-tRNA, so the 60 S subunit creates the 80 S initiation complex.

Polypeptide Chain Termination

• Polypeptide release factor complexed with GTP binds to the stop codon and uncouples peptidyltransferase activity, so the peptide chain is transferred to water (energy cost = 1 GTP)
• Upon polypeptide release, the ribosome dissociates into subunits, so eukaryotes have one release factor; prokaryotes have two.

Polyribosomes

• Also known as polysomes, allowing synthesis of several polypeptides concurrently on the same mRNA molecule
• It occurs in both prokaryotes and eukaryotes.

Posttranslational Modification

• Polypeptides undergo a variety of modifications to become biologically active.
• Trimming: Amino-terminal methionine and carboxyterminal amino acids are removed.
• Proteolytic Processing: It converts inactive, stored precursor forms into an active form.
• Signal Sequences (Targeting Sequences): It directs newly synthesized proteins to their ultimate location, then removed by special peptidases.
• Prenylation: Isoprenyl groups attach to cysteine side chains.
• Glycosylation: One or more carbohydrates or oligosaccharides are added to specific amino acid side chains.

Cellular Sorting of Proteins

• Cells that produce proteins for secretion and functions in various subcellular organelles.
• Signal sequences for secreted proteins
• Mannose phosphate tagging for lysosomal enzymes
• Translocase presequences for mitochondrial proteins
• Nuclear localization sequences for nuclear proteins.

Secreted Proteins

• Signal sequence targets a polypeptide to the ER lumen
• A ribonucleoprotein signal recognition particle (SRP) is recognized and bound during synthesis's early stages, so polymerization stops and SRP guides the ribosome to a membrane receptor on the ER
• The growing polypeptide passes through a translocon channel in the membrane into the lumen, so the signal sequence is then removed.

Lysosomal Enzymes

• Enzymes are tagged by a phosphotransferase enzyme in a two-step reaction
• Phosphate is attached to terminal mannose residues on oligosaccharides of mannose-rich glycoproteins
• Mannose 6-phosphate binds to a receptor in the Golgi membrane, so vesicles containing the receptor-bound enzymes bud off the Golgi to fuse with lysosomes.

Mitochondrial Proteins

• Most total proteins in the mitochondrion are proteins synthesized in the cytoplasm from nuclear genes, rather than the mitochondrial genome
• Mitochondrial proteins are imported by translocation through translocation complexes, so proteins must be unfolded and passed through the membrane in a single strand, then refolded in the matrix.

Proteins Destined for the Nucleus

• Including histones and a nuclear localization signal with positively charged amino acids.
• A carrier protein, called an importin, binds and imports through the nuclear pores.
• After translocation, nuclear proteins are transported in a folded state.

Translational Repression

• Messenger RNA (mRNA) is not always translated when it appears in the cytoplasm
• Translation repressor proteins can bind to stem-loop structures near the 5' end of the mRNA, preventing initiation complex formation.
• Ferritin is an iron storage protein for states with excess iron, so it binds and dissociates when iron levels are restored.

Unfertilized Ovum

• Filled with ribosomes and mRNA, without polyribosomes, so translation begins upon fertilization.

Protein Degradation

• Produces AA for synthesis of new proteins, where cellular proteins use the ubiquitin-proteasome system
• Proteins are labeled with ubiquitin through covalent attachment to a lysine side chain, and their amino terminus composition determines how quickly they are ubiquinated
• Protein degradation is then labelled with polyubiquination, which increases turnover/degradation, entering a barrel-shaped proteosome complex
• The tagged proteins are then cut into small peptide fragments, digested to amino acids by proteases in an energy-requiring process.