Translation

• RNA is a single-stranded polynucleotide with a sugar (ribose)-phosphate backbone and 4 different bases (A,G,C,U)
• Complementary base-pairing properties


  G pairs with C, and A pairs with U
• Types of RNAs


  • mRNAs
  • tRNAs
  • rRNAs
  • ncRNAs
• mRNA is a molecule that specifies the amino acid sequences of a protein
• RNA polymerase


  • Enzyme that carries out transcription, holoenzyme
  • Bacteria contain a single type of RNA polymerase
  • Eukaryotes have three RNA polymerases—polymerase I, II, and III
• PROMOTOR


  Nucleotide sequence in DNA to which RNA polymerase binds to start transcription
• TERMINATOR


  Signals the end of transcription
• Eukaryotic mRNA is synthesized by RNA polymerase II
• RNA polymerase II requires general transcription factors to initiate transcription on its chromatin template inside the cell
• During the elongation – termination phase of transcription


  A special nucleotide is added to the 5´ end (capping), intron sequences are removed (splicing), and the 3´ end is generated (cleavage and polyadenylation)
• For some genes, RNA is the final product, usually transcribed by RNA polymerase I or RNA polymerase III in eukaryotes
• Translation


  The genetic code is read consecutively in groups of three nucleotides
• 64 possible combinations of three nucleotides in the genetic code
• Most amino acids are encoded by more than one codon, except Methionine and Tryptophan
• STOP CODONS do not specify any amino acid but signal the end of the protein-coding sequence
• AUG is the initiation codon, signaling the start of a protein coding message and specifying methionine
• Reading frame


  A way of dividing the sequence of nucleotides in a nucleic acid molecule into a set of triplets
• Transfer RNAs (tRNAs)


  • Molecules that match amino acids to codons in mRNA
  • Approximately 80nt in length
  • Contain an anticodon that pairs with the complementary codon in mRNA
  • The amino acid is attached at the 3´ end of the tRNA
  • T-loop acts as a recognition site for the ribosome to form a tRNA-ribosome complex
• Anticodon


  • A set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule
• Attaching amino acid to tRNA

  The amino acid matching the codon/anticodon pair is attached at the 3´ end of the tRNA (ACC)
• 1. loop


  • Acts as a special recognition site for the ribosome to form a tRNA-ribosome complex during translation
• 1. loop


  • Site of recognition for aminoacyl t-RNA synthetase
• tRNA structure


  • Undergoes further folding to form a compact L-shaped structure held together by additional hydrogen bonds between different regions of the molecule
• Some tRNAs bind to multiple codons (Wobble)
• Loading a tRNA with an amino acid

  1. Aminoacyl-tRNA synthetases covalently couple each amino acid to its appropriate set of tRNA

  2. Different synthetase enzyme for each amino acid (20 synthetases in all)

  3. Aminoacyl-tRNA synthetases recognize tRNAs mainly by interacting with the anticodon loop
• tRNA editing


  1. The synthetase must select the correct amino acid through a two-step recognition mechanism (tRNA editing)

  2. Accurate discrimination between two similar amino acids forces the amino acid into a second pocket in the synthetase

  3. Once an amino acid enters this editing pocket, it is hydrolysed from the AMP and released from the enzyme
• Incorporation of an amino acid into a protein

  1. A protein is synthesized from its N-terminal end to its C-terminal end

  2. Protein synthesis occurs by forming a peptide bond between the carboxyl group at the end of a polypeptide chain and a free amino group on an incoming amino acid

  3. The peptidyl-tRNA linkage that activates the growing end is regenerated during each addition
• Ribosomes


  • The Nucleolus is a ribosome-producing factory
  • Site for the processing of rRNAs and their assembly into ribosomes
  • Not bound by a membrane
  • Large aggregate of rRNA genes, precursor rRNAs, mature rRNAs, rRNA processing enzymes, snoRNPs, ribosomal protein subunits, and partly assembled ribosomes
  • Has two subunits, each with rRNA and many other proteins
  • Large subunit 60S contains 3 RNAs: 28S, 5,8S, and 5S and approx. 50 proteins
  • Small subunit has 18S RNA and approx. 30 proteins
  • The small subunit provides a framework on which the tRNAs can be accurately placed
• Ribosomes


  • Has two subunits, each with rRNA and many other proteins
  • Large subunit 60S contains 3 RNAs: 28S, 5,8S, and 5S, and approx. 50 proteins
  • Small subunit has 18S RNA and approx. 30 proteins
  • Small subunit provides a framework for tRNAs to be accurately matched to mRNA codons, while the large subunit catalyses the formation of peptide bonds linking amino acids into a polypeptide chain
• RNA-binding sites in the ribosome


  • Each ribosome has three binding sites for tRNA: A-, P-, and E-sites, and one binding site for mRNA
• Translating an mRNA molecule

  Three-step cycle: 1. Aminoacyl-tRNA molecule binds to a free A-site on the ribosome 2. New peptide bond is formed 3. mRNA moves three nucleotides through the small-subunit chain, ejecting the tRNA molecule
• Translation initiation (eucaryotes)


  AUG-start codon, Initiator tRNA always carries methionine (Met-tRNAi), Eucaryotic Initiation Factors (eIFs)
• Translation initiation (bacteria)


  Polycistronic mRNA encodes several different proteins
• Translation elongation


  mRNA is translated from 5’-3’ direction, protein from N-terminal to C-terminal, Two elongation factors: EF-Tu and EF-G in bacteria, EF1 and EF2 in eucaryotes
• Translation termination


  Stop codons (UAA, UAG, UGA), Release factors bind to any ribosome with a stop codon (site A), forcing the peptidyl transferase to catalyze the addition of a water molecule
• Polyribosomes


  • Group of ribosomes bound to an mRNA molecule
• Antibiotics kill bacteria by inhibiting bacterial protein synthesis. Different antibiotics bind to different regions of bacterial ribosomes
• Nonsense-mediated mRNA decay (NMD)
• Protein folding


  Chaperones help guide the folding of many proteins (Hsp70), Chaperones help guide the folding of many proteins (Hsp60)
• Protein degradation


  Proteasome is present in the cytosol and the nucleus. Active sites: central cylinder, 19S cap, UPS (Ubiquitin Proteasome System)