Gene expression: translation, including defects
translation, antibiotics

the genetic code

  • The genetic code: converts mRNA codons into amino acids.
    • Codons are triplets: coded by 3 nucleotides.
      • Start codon: AUG (Augment means to increase), codes for methionine.
      • Stop codons: UGA, UAA, UAG (U Go Away, U Are Away, U Are Gone)
    • Commaless: read without gaps.
    • Nonoverlapping.
    • Universal: almost all organisms use the same code.
    • Degenerate: More than one codon per amino acid.
      • Wobble hypothesis: molecular mechanism for codon degeneracy is that the third letter of the codon can wobble in its bonding with the anticodon.
  • Amino Acids
    amino acids

structure and function of tRNA

  • tRNA structure: cloverleaf secondary structure, L-shaped tertiary structure.
    • Tip contains the anticodon.
    • 3'- ends in CCA: the amino acid is attached to the 3'-OH of the A.
  • tRNA function: bring the correct amino acid to the correct codon.
    1. Charging a tRNA: aminoacyl-tRNA synthetase attaches the correct amino acid to the correct tRNA. Specificity determined by tRNA structure interaction with aminoacyl-tRNA synthetase.
    2. tRNA brings the correct amino acid to the correct codon. Specificity determined by codon-anticodon interaction.

structure and function of ribosomes

  • Ribosome structure:
    • Prokaryotic: 70S = 30S + 50S
      • Small subunit: 30S = 16S rRNA + proteins.
      • Large subunit: 50S = 23S rRNA + proteins + 5S rRNA.
    • Eukaryotic: 80S = 40S + 60S
      • Small subunit: 40S = 18S rRNA + proteins.
      • Large subunit: 60S = 28S rRNA + proteins + 5S rRNA + 5.8S rRNA.
        • Ricin/abrin/α-sarcin inactivates 28S rRNA.
  • Ribosome function: protein synthesis

protein synthesis

  • Prokaryotic:
    1. Initiation:
      • Shine-Dalgarno sequence on mRNA bind small subunit. tRNA f-met bind start codon on mRNA at the P site.
      • Initiation factors (IFs) help bring all the above together. IF-1 and IF-3 helps mRNA binding. IF-2 + GTP helps tRNA binding.
      • IFs dissociate, large subunit comes along and caps over the small subunit, sandwiching mRNA and tRNA in between.
      • Aminoglycosides affect initiation and causes mRNA misreading.
        • Paromomycin: increases error rate.
        • Streptomycin: inhibits initiation.
    2. Elongation:
      • Decoding: aminoacyl-tRNA's anticodon matches mRNA codon at the A site. EF-Tu + GTP help bring aminoacyl-tRNA to the A site. EF-Ts recharges EF-Tu with GTP.
        • Tetracycline inhibits aminoacyl-tRNA binding.
      • Transpeptidation: Peptide bond forms from the N-term of A site amino acid attacks the C-term of the P site chain. Results in chain migrated to A site. The reaction is catalyzed by the rRNA of the large subunit.
        • Puromycin is an analogue of tyr-tRNA with a dead C-term (amide rather than ester), causes premature termination.
        • Chloramphenicol inhibits peptidyl transferase in prokaryotic large subunit.
      • Translocation: the tRNAs along with the mRNA moves down the ribosome. The previous P site tRNA enters E site (exits), and the previous A site tRNA (holding the chain) moves to the P site. The A site is now empty and ready to accept the next tRNA. EF-G + GTP needed.
        • Erythromycin inhibits prokaryotic translocation in large subunit.
        • Fusidic acid affects EF-G (prevents it from dissociating from large subunit).
        • Macrolides bind 50S, inhibit translocation.
        • Clindamycin binds 50S, inhibit translocation.
    3. Termination:
      • Release factor 1 (RF1) or RF2 binds stop codon.
      • Hydrolysis of ester bond between C-term of protein chain and 3'-tRNA.
      • RF3-GTP knocks off RF1 or RF2, then dissociates itself.
      • Ribosomal recycling factor (RRF) and EF-G + GTP knocks off the tRNA, the mRNA, and then dissociate themselves.
  • Eukaryotic: similar to prokaryotic, with the most difference in initiation.
    1. Initiation:
      • initator tRNA-met binds to small subunit by initiation factors at the P site.
      • 5'-cap and 5'-UTR of mRNA binds to small subunit by initiation factors.
      • tRNA anticodon slides along mRNA until it finds AUG start codon.
      • IFs dissociate, large subunit comes along and caps over the small subunit, sandwiching mRNA and tRNA in between.
      • Eukaryotes have a whole lot more IFs than prokaryotes.
    2. Elongation:
      • Decoding: aminoacyl-tRNA's anticodon matches mRNA codon at the A site. eEF1A (Eukaryotic equivalent of EF-Tu) and eEF1B (Eukaryotic equivalent of EF-Ts) needed.
      • Transpeptidation: formation of peptide bond, where A site amino acid N-term attacks P site chain's C-term.
        • Puromycin mimics tyr-tRNA but has a dead C-term, and causes premature termination because transpeptidation can't occur.
        • Cycloheximide inhibits peptidyl transferase on eukaryotic large subunit.
      • Translocation: tRNA, mRNA slides down ribosome. P (tRNA) → E, A (tRNA + chain) → P, A site now empty. eEF2 (Eukaryotic equivalent of EF-G) needed.
        • Diphtheria toxin inactivates eEF2.
    3. Termination:
      • eRF1 (Eukaryotic release factor) binds stop codon.
      • Hydrolysis of ester bond between C-term of protein chain and 3'-tRNA.
      • eRF3 (Eukaryotic equivalent of RF3) knocks off eRF1, then dissociates.
      • Eukaryotic equivalent of RRF and eEF2 involved in knocking off mRNA and tRNA (not well understood).

regulation of translation

  • Regulation of gene expression in prokaryotes is predominantly transcriptional control. Although, differences in Shine-Dalgarno sequences play a minor role in translational control.
  • Eukaryotes have regulation of translation because their mRNA have a much longer life time.
  • Eukaryotic initiation factors.
    • Reticulocytes translate hemoglobin when there's heme present (normal eIF2), but stops translating hemoglobin when heme is scarce (phosphorylated eIF2).
      • Heme-regulated inhibitor (HRI) is the kinase.
      • eIF2 phosphatase reverses the action of HRI.
    • Interferons inhibits translation of viral proteins.
      • Interferons induce an eIF2 kinase (PKR/DAI), which is activated upon binding to viral dsRNA. Many viruses express PKR inhibitors.
      • Interferons also induce mRNA degradation (RNase L activated by presence of dsRNA).
    • PKR-like endoplasmic reticulum kinase (PERK) phosphorylates eIF2 when there's too much unfolded proteins in the ER. This prevents the cell from being overwhelmed and damaged.
      • Wolcott-Rallison syndrome: defective PERK leads to pancreatic β cells, and causes type I diabetes and multiple systemic disorders.
    • Cap-binding protein (eIF4E)
      • More active when phosphorylated.
      • Binding of inhibitors inactivates cap-binding proteins.
  • mRNA masking: binding of mRNA to proteins prevent them from being translated.
    • mRNA in oocyte is masked by formation of ribonucleoporotein particles (RNPs).
    • Regulate gene expression in early development by unmasking the stored RNPs.
  • Cytoplasmic polyadenylation
    • Normally, polyA is added in nucleus, but cytoplasmic polyA addition regulates translation on another level.
  • Antisense oligonucleotides:
    • Antisense hybridizes with mRNA, forms dsRNA that is target for RNase H.
    • RNase H cleaves mRNA but leaves antisense (chemically modified, resistant to RNase)
    • Antisense can go hybridize again.

post-translational modifications, including phosphorylation, addition of CHO units

  • Cleavage:
    • Remove initiator met or f-met.
    • Remove signal peptide (initially used to target the RER).
    • Zymogen (proenzyme) → active enzyme.
      • -sinogen → -sin. Eg. Pepsinogen → pepsin.
    • Procollagen → collagen
      • Ehlers-Danlos syndrome VII: mutation in procollagen that prevents it from being cleaved.
    • Polyproteins → protein components.
  • Modification:
    • Hydroxylation of procollagen chain so that the mature collagen triple helix can hydrogen bond.
    • Glycosylation to make glycoproteins by adding sugar residues.
      • Phosphorylation of mannose targets protein to lysosomes.
      • Complex carbohydrates targets protein to cell membrane.
      • Dolichol transfers initial carbohydrate chain onto emerging peptide. Then all but one glucose residue (recognized by chaperones) is removed.
      • Chaperones like calreticulin (CRT), calnexin (CNX), and BiP prevent aggregation of unfolded glycoprotein. Misfolded proteins are degraded.
    • Phosphorylation to activate or inactivate enzymes.
    • Acetylation. Eg. Histone acetylation activates genes.
    • Methylation. Eg. Histone methylation restricts histone deacetylation.
  • Splicing:
    • Intein cut out.
    • Exteins spliced together.
    • Inteins are homing endonucleases, cut inteinless extein genes, then insert intein gene (like molecular parasite, mechanism of insertion is by recombination with homologous intein-containing extein gene).

protein degradation

  • Targets for degradation:
    • Random degradation responsible for turnover of proteins.
    • Abnormal proteins are degraded.
    • Proteins that are involved in cell-cycle control (eg. cyclins).
    • Protein degradation goes up when cell is starved to provide energy.
  • Mechanisms:
    • Ubiquitin tags protein for degradation.
      • Ubiquitin (analogous to C-term) forms amide bond with N-term of protein.
      • E1, E2, E3 helps ubiquitin to bond with protein.
      • Protein degraded inside the proteosome (barrel) with ATP hydrolysis.
      • Ubiquitin is not degraded by the proteosome.
    • Lysosomes: fusion with either autophagic vacuoles or with endocytosed vacuoles.
      • Diabetes mellitus stimulates lysosomal degradation of proteins.
      • Muscle wastage.
      • Rheumatoid arthritis.