Gene translation

  • tRNA
    • Structure
      • 75–90 nucleotides, 2º structure, cloverleaf form, anticodon end is opposite 3′ aminoacyl end.
      • All tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along with a high percentage of chemically modified bases. CCA Can Carry Amino acids.
      • The amino acid is covalently bound to the 3′ end of the tRNA.
      • T-arm: contains the TΨC (ribothymidine, pseudouridine, cytidine) sequence necessary for tRNA ribosome binding.
        • T-arm Tethers tRNA molecule to ribosome.
      • D-arm: contains dihydrouridine residues necessary for tRNA recognition by the correct aminoacyl-tRNA synthetase.
        • D-arm Detects the tRNA by aminoacyl-tRNA synthetase.
      • Acceptor stem: the 5′-CCA-3′ is the amino acid acceptor site.
    • Charging
      •  Aminoacyl-tRNA synthetase (1 per amino acid; “matchmaker”; uses ATP) scrutinizes amino acid before and after it binds to tRNA. If incorrect, bond is hydrolyzed. The amino acid-tRNA bond has energy for formation of peptide bond. A mischarged tRNA reads usual codon but inserts wrong amino acid.
      • Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for accuracy of amino acid selection.
      • ATP—tRNA Activation (charging).
  • Protein synthesis
    • Initiation
      • Eukaryotic initiation factors (eIFs) identify either the 5′ cap or an internal ribosome entry site (IRES). IRES can be located at many places in an mRNA (most often 5′ UTR).
      • The eIFs then help assemble the 40S ribosomal subunit (responsible for binding mRNA [the protein synthesis template] and tRNA [carries amino acids]) with the initiator tRNA and are released when the mRNA and the ribosomal 60S subunit (contains peptidyl transferase, the enzyme that catalyzes peptide bond formation between amino acids) assemble with the complex.
      • Requires GTP.
      • Eukaryotes: 40S + 60S → 80S (Even).
      • PrOkaryotes: 30S + 50S → 70S (Odd).
      • Small ribosomal subunit (40s or 30s) binds to the 5′ cap of mRNA for AUG (methionine start sequence)
      • Synthesis occurs from N-terminus to C-terminus.
      • In prokaryotes, the Shine-Delgarno sequence is located upstream from initiation codon AUG, which recognizes N-formylmethionine-tRNA (forming the prokaryotic 30s initiation complex).
      • In eukaryotes, the Kozak consensus sequence is analogous to the Shine-Delgarno sequence in E coli. It is defined by (gcc)gccRccAUGG, in which R is either adenine (A) or guanine (G). A purine (A or G) positioned 3 bases upstream from AUG appears to play a key role in this initiation process.
      • Polycistronic mRNA is often found in bacteria and contains multiple open reading frames that are translated into several proteins.  In contrast, eukaryotic organisms have monocistronic mRNA, which codes for only one protein.
  • Elongation
    1. Aminoacyl-tRNA binds to A site (except for initiator methionine), requires an elongation factor and GTP
    2. rRNA (“ribozyme”) catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site
    3. Ribosome advances 3 nucleotides toward 3′ end of mRNA, moving peptidyl tRNA to P site (translocation)

Think of “going APE”:

      • A site = incoming Aminoacyl-tRNA.
      • P site = accommodates growing Peptide.
      • E site = holds Empty tRNA as it Exits
  •  Termination
    • Release factor recognizes stop codon and halts translation → completed polypeptide is released from ribosome.
    • Requires GTP.
    • GTP—tRNA Gripping and Going places (translocation).
  • Posttranslational modifications
    • Trimming: Removal of N- or C-terminal propeptides from zymogen to generate mature protein (e.g., trypsinogen to trypsin).
    • Covalent alterations Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination.
  • Chaperone protein
    • Intracellular protein involved in facilitating and/or maintaining protein folding. For example, in yeast, heat shock proteins (e.g., HSP60) are expressed at high temperatures to prevent protein denaturing/misfolding.

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