Lecture 8

• Translation

  The cellular process that converts genetic information in the form of mRNA into a specific sequence of amino acids to form proteins
• Key Components of Translation

  • mRNA: Serves as the template for protein synthesis
  • tRNA: Transfers specific amino acids to the ribosome during protein synthesis
  • Ribosomes: Catalyze the formation of peptide bonds between amino acids to form polypeptides
  • Aminoacyl-tRNA synthetase
• Initiation

  1. Assembly of the ribosomal subunits, mRNA, and the initiator tRNA at the start codon of the mRNA

  2. Initiation factors play crucial roles in ensuring that translation begins at the correct start codon
• Elongation

  1. Amino acids are sequentially added to the growing polypeptide chain

  2. Each tRNA brings a specific amino acid to the ribosome, matching its anticodon to the codon on the mRNA

  3. Energy in the form of GTP is used to facilitate peptide bond formation and the translocation of the ribosome along the mRNA
• Termination

  1. When a stop codon is reached, the ribosome releases the polypeptide and dissociates from the mRNA

  2. Release factors help terminate translation and release the completed polypeptide from the ribosome
• Translational Control

  Cells regulate the efficiency and rate of translation in response to environmental conditions and developmental cues, affecting protein synthesis globally or specifically
• Ribosome Structure

  • Composed of two subunits, each containing rRNA and proteins
  • The ribosome reads mRNA codons and catalyzes peptide bond formation
• tRNA Role

  • tRNAs adapt genetic information by matching their anticodon regions to corresponding codons on the mRNA, while carrying the appropriate amino acid
• Aminoacyl tRNA Synthetases

  Enzymes that charge tRNAs with their correct amino acids in an ATP-dependent manner
• Translation is a target for antibiotic therapy in bacteria, as bacterial ribosomes are sufficiently different from eukaryotic ribosomes, allowing for selective inhibition
• Misregulation of translation can lead to diseases, including cancer and metabolic disorders
• Large Subunit of the Ribosome

  • Consists of over 64,000 atoms
  • Plays a crucial role in the catalytic processes of translation, such as peptide bond formation
• Small Subunit of the Ribosome

  • Composed of more than 29,000 atoms
  • Primarily involved in decoding the mRNA
• rRNAs

  • The ribosome includes essential RNA components like the 16S rRNA in bacteria, which have highly conserved regions critical for function and variable regions that are more tolerant to mutations
• Initial sequencing of the E. coli rRNAs in 1978 has led to advanced models based on conserved base pairing, showing the robust framework that supports ribosomal function
• The conservation of rRNA structure across species emphasizes the evolutionary importance of ribosomal architecture. It includes Watson-Crick base pairing that is maintained across species, highlighting essential functional regions
• Peptidyl Transferase Center (PTC)

  • Located in the large subunit, this is the enzymatic heart of the ribosome, where peptide bond formation occurs
  • It acts as a ribozyme, with key nucleotides like A2486 playing pivotal roles in catalysis
• Functional Dynamics of the PTC

  • Aligns the aminoacyl tRNA in the A site and the peptidyl tRNA in the P site for efficient peptide bond formation
  • The positioning ensures the correct transfer of the growing peptide chain
• Polypeptide Exit Tunnel

  • Structure and Role: The tunnel within the large subunit through which the growing polypeptide exits
  • It is crucial for the nascent peptide to fold partially into secondary structures like alpha-helices even before emerging fully from the ribosome
  • Distance from Catalytic Site: The closest ribosomal protein (L3) is located too far (18.4 Å) to directly participate in the catalytic process, underscoring the ribozyme nature of the ribosome where RNA plays a more active role than proteins in catalysis
• tRNA Binding Sites in the Ribosome

  • A Site (Acceptor): Where incoming aminoacyl tRNA binds
  • P Site (Peptidyl): Holds the tRNA carrying the growing polypeptide chain
  • E Site (Exit): Where deacylated tRNA resides before exiting the ribosome
• Many antibiotics exert their effects by targeting bacterial ribosomes, exploiting subtle structural differences between bacterial and eukaryotic ribosomes
• The conservation of ribosomal RNA structures across different organisms highlights their fundamental role in life's machinery and provides insights into evolutionary biology
• tRNAs

  • Named for the amino acids they carry, e.g., tRNA^Ala for alanine
  • Each tRNA molecule is charged with a specific amino acid, a process catalyzed by enzymes called aminoacyl-tRNA synthetases
  • Isoaccepting tRNAs: Multiple tRNAs can carry the same amino acid but may have different anticodon sequences, enhancing the flexibility and efficiency of protein synthesis
• tRNA Structure - Cloverleaf Model

  • General Structure: tRNA molecules fold into a characteristic L-shaped three-dimensional structure that resembles a cloverleaf in 2D representations
  • Important Features: Anticodon Loop, D Loop, TψC Loop, CCA Tail, Modified Nucleosides
• Aminoacyl-tRNA Synthetases

  • Enzymes responsible for the accurate attachment of amino acids to their corresponding tRNAs
  • The enzyme's specificity ensures that the genetic code is accurately translated into proteins
• Shared Reactions of tRNAs

  • Interactions with Elongation Factors
  • Binding to the Ribosome's A Site
  • CCA Terminal Addition
  • Invariant Modifications
• Unique Reactions of tRNAs

  • Aminoacylation by Synthetases
  • Codon-Anticodon Interaction
  • Recognition of Initiator tRNA
• Aminoacyl-tRNA Synthetases

  • Enzymes essential for protein synthesis
  • They charge tRNAs with their respective amino acids, ensuring the correct amino acid is added to the growing peptide chain during translation
• Diversity and Structure of Aminoacyl-tRNA Synthetases

  • Vary significantly in size (40 to 100 kDa) and may be monomeric, dimeric, or tetrameric
  • Classified into two classes, each consisting of ten enzymes
• Catalytic Sites of Aminoacyl-tRNA Synthetases

  • Class I: The catalytic site is at the N-terminal, arranged in an α-β-α-β fold
  • Class II: The catalytic site is centrally located, characterized by a β-sheet surrounded by α-helices
• tRNA Identity Elements

  Specific nucleotide sequences or structural features within the tRNA that are recognized by the synthetases, crucial for accurate aminoacylation
• Editing Mechanism of Synthetases

  • Proofreading: Aminoacyl-tRNA synthetases can proofread to ensure high fidelity in protein synthesis
  • Initial Selection: Rejects amino acids that are too large for the activation site
  • Hydrolytic Editing: Smaller, incorrectly charged amino acids are hydrolyzed at the editing site
• Mutations or dysfunctions in aminoacyl-tRNA synthetases are linked to various diseases, making them potential targets for drug development
• Mechanisms of Action of Antibiotics Targeting Ribosomes


  • Antibiotics like erythromycin and chloramphenicol target the peptidyl transferase activity of ribosomes, crucial for peptide bond formation during protein synthesis
  • They bind directly to the peptidyl transferase center of the 23S rRNA in prokaryotes
  • Other antibiotics, such as streptomycin and tetracycline, inhibit different stages of translation, such as initiation and the binding of aminoacyl-tRNAs to the ribosome
• Bacterial Defense Mechanisms

  • Colicin E3: A protein that inhibits the growth of bacterial cells lacking the Col plasmid by cleaving 16S rRNA, disrupting ribosomes' ability to initiate protein synthesis
  • Antibiotic Resistance: Bacteria can evolve resistance through genetic mutations or by acquiring resistance genes, which can degrade or modify antibiotics, change the antibiotic target, or pump the antibiotic out of the cell
• Ribotoxins


  • Ricin and α-sarcin: Toxins that target the eukaryotic ribosome by inactivating key structures in the 28S rRNA
  • Ricin removes a single adenine from a conserved loop, blocking elongation factor interaction, which is essential for translation
  • α-sarcin cleaves a single bond in the rRNA, leading to inhibition of protein synthesis
• Common Antibiotics and Their Targets

  • Aminoglycosides (e.g., Streptomycin): Inhibit initiation and cause misreading of mRNA in prokaryotes
  • Tetracycline: Binds to the 30S subunit and prevents aminoacyl-tRNA binding, crucial for translation progression
  • Puromycin: Mimics the aminoacyl part of tRNA and causes premature chain termination
• The specific interactions of these antibiotics with the ribosomal machinery provide valuable insights into designing new drugs that can target bacterial ribosomes without affecting eukaryotic cells
• Understanding the structural basis for the specificity and mechanism of action can aid in developing antibiotics that bacteria are less likely to resist
• Research into how antibiotics interact with the ribosome informs the development of novel antibiotics to combat resistant bacterial strains