Protein Primary Structure and Translation

Hierarchy of Protein Structure

Primary (1°) Structure: Defined as the specific linear sequence of amino acids in a polypeptide chain.
    * It determines all higher-level structural characteristics of the protein.
    * Properties are dictated by the chemistry of the lateral R groups.
    * Possesses directionality, moving from the N-terminus to the C-terminus.
    * Has a specific length (a set number of amino acids per protein).
Secondary (2°) Structure: Refers to local folding of the polypeptide chain into repeating units, such as helices or sheets.
Tertiary (3°) Structure: The full folding of a single polypeptide chain into its comprehensive three-dimensional structure.
Quaternary (4°) Structure: The association and spatial arrangement of multiple polypeptide subunits into a larger protein complex.

Information Flow and Genomic Correlation

Central Dogma: Information flows from DNA to RNA and finally to protein.
DNA Strands:
* Coding Strand: The DNA strand that has the same sequence as the mRNA (except T instead of U), moving 5' to 3'.
* Template Strand: The DNA strand complementary to the mRNA, moving 3' to 5'. The mRNA is synthesized by copying this specific strand.
mRNA Components:
    * Untranslated Section: Sequences at the beginning of the mRNA that do not code for amino acids.
    * Initiation Signal: Specific sequences that mark the start of translation.
    * Codons: Triplets of nucleotides that correspond to specific amino acids.
Protein Synthesis specifics:
* The primary structure (sequence) of the protein corresponds directly to the gene sequence in the DNA.
* Methionine Processing: Methionine (Met) is the initial amino acid in eukaryotes and many bacteria; however, it is often cleaved during or after translation in various proteins.

Mechanics of Translation

Definition: Translation is the mRNA template-directed biosynthesis of proteins.
Key Molecular Components:
    * Codon: A sequence of 3 consecutive nucleotides on mRNA specifying a single amino acid.
    * Anticodon: A complementary sequence located on tRNA that pairs with the mRNA codon.
    * Ribosome: A large nucleoprotein machine that facilitates the interaction between mRNA and tRNAs to build the polypeptide chain.
    * Direction of Travel: The ribosome moves along the mRNA in the 5' to 3' direction.
The Genetic Code:
    * Redundancy: The code is degenerate, meaning multiple codons can code for the same amino acid.
    * Wobble Base: The third position of many codons allows for non-standard pairing, providing flexibility.
    * Start Codon: AUG always codes for Methionine (Met) and establishes the essential reading frame.
    * Stop Codons: These signal the end of translation and do not code for any amino acid.
    * Shine-Dalgarno Sequences: In bacteria, these sequences help align the mRNA on the ribosome to ensure translation begins at the correct AUG start codon.

tRNA Charging and Ribosome Composition

Aminoacyl-tRNA Synthetases: These enzymes "charge" tRNAs by attaching the appropriate amino acid.
    * Activation Step: The amino acid is activated by ATP to form an aminoacyl adenylate on the $COO^-$ group. Inorganic pyrophosphate ( $PP_i$ ) is released.
    * Thermodynamics of PPi: The hydrolysis of $PP_i <br>ightleftharpoons 2P_i$ yields $ext{-}19.220194451006528 imes 10^3 ext{ J mol}^{-1}$ (or $-19 ext{ kJ mol}^{-1}$ ).
    * Coupling: The activated amino acid is then coupled to the 3' end of the tRNA, and AMP is released.
The Ribosome (Nucleoprotein Complex):
    * Composition: Approximately 65 ext{%} RNA and 35 ext{%} protein.
    * Bacterial Abundance: E. coli cells can contain up to $20,000$ ribosomes, accounting for 25 ext{%} of the dried cell mass.
    * Catalytic Efficiency: They catalyze peptide bond formation at a rate of $12$ to $21$ amino acids per second at $37^ ext{ ∘}C$ .

Stages of Translation

Soluble Protein Factors

Initiation Factors (Bacteria):
    * IF1: Promotes dissociation of the 70S ribosome into subunits ( $IF1$ and $IF3$ prevent re-association).
    * IF2: A GTPase; helps attach the initiator tRNA.
    * IF3: Prepares mRNA for ribosome binding.
Elongation Factors (Bacteria):
    * EF-Tu: A GTPase; delivers aminoacyl-tRNA to the A site.
    * EF-Ts: Recharges EF-Tu with GTP.
    * EF-G: A GTPase; facilitates translocation (movement of ribosome).
    * EF-P: Assists in the translation of consecutive Proline codons.
Termination Factors (Bacteria):
    * RF1: Recognizes UAA and UAG stop codons.
    * RF2: Recognizes UAA and UGA stop codons.
    * RF3: A GTPase that promotes the release of the components.

Stage 1: Initiation

Bacterial Ribosome (70S): Composed of a large 50S subunit and a small 30S subunit (S stands for Svedberg units, measuring sedimentation rate).
Process:
    1. IF1 and IF3 bind to the 30S subunit to prevent association with 50S.
    2. mRNA and the initiator tRNA (carrying Met) bind the 30S subunit, assisted by IF2-GTP.
    3. The 50S subunit binds to the 30S initiation complex.
    4. IF2 hydrolyzes its bound GTP, and all initiation factors dissociate.
    5. The initiator tRNA is positioned in the P site.

Ribosomal Binding Sites

A Site (Aminoacyl): Accepts the incoming aminoacyl-tRNA.
P Site (Peptidyl): Holds the tRNA attached to the growing polypeptide chain.
E Site (Exit): Holds the empty tRNA before it leaves the ribosome.

Stage 2: Elongation

Process:
    1. The aminoacyl-tRNA corresponding to the next mRNA codon binds to the A site with help from EF-Tu-GTP.
    2. Peptide Bond Formation: The amine group of the amino acid in the A site attacks the carboxylate of the amino acid/chain attached to the tRNA in the P site. This transfers the polypeptide to the tRNA in the A site.
    3. Translocation: The ribosome moves. The tRNA from the P site moves to the E site, and the peptidyl-tRNA moves from the A site to the P site. This requires EF-G-GTP.
    4. The empty tRNA exits from the E site, and the next codon is exposed in the A site.

Stage 3: Termination

Process:
    1. A stop codon enters the A site. Because there is no corresponding tRNA, a release factor (RF1 or RF2) binds instead.
    2. A water molecule performs a nucleophilic attack on the peptidyl-tRNA, releasing the completed polypeptide chain.
    3. RF3 stimulates the release of the factors through GTP hydrolysis.
    4. The empty tRNA exits, and IF1/IF3 stimulate the dissociation of the 50S and 30S subunits from the mRNA for recycling.

Energetics and Antibiotic Inhibition

Energetic Cost: Translation is extremely expensive. For a protein of $100$ amino acids:
    * Initiation: $1$ GTP (IF2).
    * Elongation (per bond): $1$ ATP (charging) + $1$ GTP (decoding/EF-Tu) + $1$ GTP (translocation/EF-G). Total for 99 bonds is $3 imes 99 = 297$ .
    * Correction: The charging tRNA step yields $AMP + PP_i$ , which counts as $2$ high-energy bonds because of subsequent $PP_i$ hydrolysis. Calculation: $1 + ((1+1+2) imes 99) + 1 = 398$ high-energy phosphate compounds.
Antibiotic Inhibitors:
    * Tetracycline: Inhibits aminoacyl-tRNA binding to the A site.
    * Streptomycin: Causes misreading of codons by interfering with tRNA-mRNA pairing.
    * Erythromycin: Binds the 23S rRNA and blocks translocation (elongation).
    * Chloramphenicol: Competitive inhibitor of the peptidyltransferase complex; its amide link resembles a peptide bond.
    * Puromycin: Mimics the 3' end of aminoacylated tRNA, enters the A site, and causes premature chain termination and release.

Mutations and Clinical Implications

Mutation Types:
    * Synonymous (Silent): No change in protein sequence due to code redundancy.
    * Non-synonymous (Missense): Changes the amino acid sequence.
        * Conservative: Substituting an amino acid with one of similar chemical properties (size, charge, hydrophobicity).
        * Nonconservative: Substituting an amino acid with one of significantly different properties.
    * Nonsense: Changes a codon to a stop codon, resulting in truncated, nonfunctional proteins.
    * Insertions and Deletions (Indels): Can delete whole amino acids or cause a frameshift, altering all downstream codons.
Myoglobin Mutation Examples:
* D4E: Aspartic Acid (D) to Glutamic Acid (E) is conservative as both are negatively charged polar amino acids.
* D53A: Aspartic Acid (D) to Alanine (A) is nonconservative as it moves from a charged polar residue to a non-polar residue.
Sickle Cell Anemia: A disease caused by a single non-synonymous mutation in the human $\beta$ -globin gene, drastically altering hemoglobin structure and function.