Lecture 9

• Translation

  A critical cellular process that converts the genetic code contained in mRNA into a sequence of amino acids, forming proteins
• Key components involved in translation

  • Ribosome
  • tRNA
  • Aminoacyl-tRNA Synthetases
  • mRNA
• tRNAfMet

  Initiates protein synthesis; formylated methionine
• tRNAMet

  Used in elongation for methionine incorporation not at the start
• Initiation

  1. Formation of a complex with the 30S ribosomal subunit, mRNA, and formylmethionine-tRNA (fMet-tRNA)

  2. Initiation factors like IF-1, IF-2, and IF-3 facilitate the assembly and stability of the initiation complex
• IF-1


  Stabilizes the 30S subunit assembly
• IF-2


  Binds fMet-tRNA in a GTP-dependent manner
• IF-3


  Prevents premature binding of the 50S subunit
• Elongation

  1. Codon Recognition

  2. Peptide Bond Formation

  3. Translocation
• EF-Tu


  Binds aminoacyl-tRNA and GTP, delivering tRNA to the ribosome. Also has a proofreading role to ensure fidelity in translation
• EF-G


  Drives translocation by moving the ribosome along the mRNA. Hydrolysis GTP to provide energy for the ribosomal movement
• Termination

  1. Occurs when the ribosome encounters a stop codon on the mRNA

  2. Release factors (e.g., RF1, RF2 in prokaryotes) bind to the ribosome, prompting the release of the newly synthesized polypeptide chain
• Summary of prokaryotic translation

  • Initiation: Formation of complex structures with specific roles for initiation factors and Met-tRNA
  • Elongation: Incorporation of amino acids into the growing peptide chain with the help of EF-Tu and EF-G
  • Termination: Release of the completed polypeptide facilitated by release factors
• Translational control

  Translation efficiency and the rate can be altered in response to cellular conditions and needs, affecting global protein synthesis
• Prokaryotic mRNA

  Often includes a Shine-Dalgarno sequence that helps align the mRNA on the ribosome, facilitating the correct initiation of protein synthesis
• Eukaryotic mRNA

  Lacks a Shine-Dalgarno sequence, relying instead on the cap structure and the poly(A) tail for ribosome recruitment and stability
• Antibiotics often target specific steps in bacterial translation, such as initiation and elongation, exploiting differences between prokaryotic and eukaryotic translation mechanisms
• Understanding these differences is crucial for the development of targeted antibiotics that do not affect eukaryotic cells
• Eukaryotic translation initiation codon

  The start codon for most eukaryotic mRNAs is AUG, which encodes for methionine
• Kozak sequence


  A consensus sequence around the AUG start codon helps in the initiation of translation, enhancing the efficiency of translation by orienting the ribosome properly
• Cap binding complex (eIF-4F)


  • eIF4E: Binds to the 5' cap of mRNA
  • eIF4A: An ATP-dependent RNA helicase that unwinds mRNA secondary structures
  • eIF4G: Acts as a scaffold for assembly of eIF4E and eIF4A, promoting the circularization of mRNA
• Ribosome assembly

  • The small ribosomal subunit (40S) is pre-bound with Met-tRNAi and scans the mRNA from the 5' end to find the start codon in the correct context
  • The large subunit (60S) joins to form the 80S initiation complex after the start codon is recognized
• Roles of translation initiation factors

  • eIF1 and eIF3: Prevent premature binding of the large ribosomal subunit
  • eIF2B: Facilitates the binding of Met-tRNAi with the small ribosomal subunit
  • eIF5: Promotes the dissociation of other initiation factors to transition to elongation phase
  • eIF6: Blocks premature association of ribosomal subunits
• Elongation

  1. Codon recognition, peptide bond formation, and translocation

  2. Elongation factors like eEF1 and eEF2 play crucial roles in peptide elongation
• Termination

  1. Occurs when a stop codon is recognized

  2. Eukaryotic release factors (eRFs) mediate the release of the polypeptide chain
• Internal ribosome entry sites (IRES)


  Enables translation initiation in the middle of mRNA, bypassing the need for the 5' cap structure, used under specific conditions like stress or viral infection
• Cell cycle and stress responses

  Translation is tightly regulated during the cell cycle and under stress, often through modifications of initiation factors
• Picornaviruses

  Use IRES to hijack the host's translation machinery, shutting off cap-dependent translation and ensuring the preferential translation of viral proteins
• Various mechanisms ensure fidelity and regulation under normal and stress conditions, highlighting the dynamic nature of cellular translation machinery
• Regulation through secondary structure changes (prokaryotes)

  Ribosome binding sites on mRNA can be temporarily blocked or exposed through alterations in the mRNA's secondary structure. This change is reversible and affects the initiation of translation depending on environmental and cellular conditions
• Autogenous regulation (prokaryotes)

  The protein product of a gene binding directly to the ribosome binding site on the same gene's mRNA. This binding inhibits further translation of the mRNA into protein, effectively regulating the amount of protein produced based on feedback from the protein itself
• Regulation through repressor proteins (eukaryotes)

  Proteins known as repressors can bind to specific sequences in the 5' untranslated region (UTR) of an mRNA. This binding can inhibit the initiation of translation by physically blocking the assembly of the ribosomal complex or altering the local RNA structure
• Differential mRNA stability (eukaryotes)

  The lifespan of mRNA molecules can vary greatly, influenced by specific sequence elements and binding proteins that determine their stability. More stable mRNA will be translated more efficiently and for longer durations, while less stable mRNA is quickly degraded, reducing protein production
• eIF2α and eIF4E phosphorylation

  The translation initiation factors eIF2α and eIF4E are critical regulatory points in eukaryotic translation. Phosphorylation of eIF2α generally reduces global protein synthesis, which is a crucial response to stress and viral infection. eIF4E phosphorylation, conversely, enhances translation and is often upregulated in cancers, making it a target for therapeutic intervention
• Autogenous control in E. coli

  The production of ribosomal proteins is highly responsive to the cell's growth rate. When growth is rapid, and ribosome demand is high, production of ribosomal proteins increases. This process is tightly linked with rRNA synthesis, ensuring a balanced production of ribosomal components
• Iron-response element binding proteins


  These proteins, such as cytosolic aconitase, can bind to iron response elements (IREs) on mRNA. Their activity is modulated by iron availability
• Excess iron


  Leads to ferritin translation for iron storage and degradation of transferrin receptor mRNA, reducing iron uptake
• Iron deficiency

  Ferritin translation is inhibited, allowing free iron to remain available for essential functions, while transferrin receptor mRNA is stabilized to increase iron uptake
• Poly(A) tail shortening

  The gradual shortening of the poly(A) tail at the end of mRNA molecules, a process that precedes mRNA decay. Loss of the poly(A) tail disrupts the interaction of mRNA with stabilizing proteins, leading to mRNA destabilization and decay
• Cleavage by endonuclease

  Specific cleavage events within the mRNA, particularly in the 3' untranslated regions (3' UTR), can trigger rapid mRNA degradation. This is an essential mechanism for controlling the levels of certain mRNAs in the cell, influencing how much protein is produced from each mRNA