Protein Synthesis Translation of the Genetic Message.pptx by pptfiles

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									Protein Synthesis: Translation of
     the Genetic Message
          Chapter Twelve
     Translating the Genetic Message
• Protein biosynthesis is a
  complex process requiring
  ribosomes, mRNA, tRNA, and
  protein factors

• Several steps are involved

• Before being incorporated into
  growing protein chain, a.a.
  must be activated by tRNA and
  aminoacyl-tRNA synthetases
 Salient features of the genetic
              code
– Triplet: a sequence of three bases (a codon) is needed
  to specify one amino acid
– Nonoverlapping: no bases are shared between
  consecutive codons
– Commaless: no intervening bases between codons
– Degenerate: more than one triplet can code for the
  same amino acid; Leu, Ser, and Arg, for example, are
  each coded for by six triplets
– Universal: the same in viruses, prokaryotes, and
  eukaryotes
              The Genetic Code


• The ribosome moves
  along the mRNA
  three bases at a time
  rather than one or
  two at a time
The Genetic Code
        • All 64 codons have
          assigned meanings

          – 61 code for amino acids

          – 3 (UAA, UAG, and UGA)
            serve as termination
            signals

          – only Trp and Met have
            one codon each
The Genetic Code

        • The third base is irrelevant
          for Leu, Val, Ser, Pro, Thr,
          Ala, Gly, and Arg

        • Amino acids coded by 2, 3,
          or 4 triplets - the third
          letter of the codon - Gly,
          for example, is coded by
          GGA, GGG, GGC, and GGU
             Wobble Base Pairing
• Some tRNAs bond to
  one codon exclusively,
  but many tRNAs can
  recognize more than
  one codon because of
  variations in allowed
  patterns of hydrogen
  bonding
   – the variation is called
     “wobble”
   – wobble is in the first
     base of the anticodon
Base Pairing Combination in the
       Wobble Scheme
If there are 64 codons, how can there
    be less than 64 tRNA molecules?
• The wobble hypothesis provides insight
  – in many cases, the degenerate codons for a given
    amino acid differ only in the third base; therefore
    fewer different tRNAs are needed because a given
    tRNA can base-pair with several codons
  – the existence of wobble minimizes the damage that
    can be caused by a misreading of the code; for
    example, if the Leu codon CUU were misread as CUC
    or CUA or CUG during transcription of mRNA, the
    codon would still be translated as Leu during protein
    synthesis
           Amino Acid Activation
• Amino acid activation
  and formation of the
  aminoacyl-tRNA take
  place in two separate
  steps
• Both catalyzed by
  aminoacyl-tRNA
  synthetase
• Free energy of
  hydrolysis of ATP
  provides energy for
  bond formation
                tRNA Tertiary Structure
• There are several recognition sites for various
  amino acids on the tRNA
                  Chain Initiation
• In all organisms, synthesis of polypeptide chain
  starts at the N-terminal end, and grows from N-
  terminus to C-terminus
• Initiation requires:
  –   tRNAfmet
  –   initiation codon (AUG) of mRNA
  –   30S ribosomal subunit
  –   50S ribosomal subunit
  –   initiation factors IF-1, IF-2, and IF-3
  –   GTP, Mg2+
• Forms the initiation complex
The Initiation Complex
             Chain Initiation
– tRNAmet and tRNAfmet contain the triplet 3’-UAC-5’
– Triplet base pairs with 5’-AUG-3’ in mRNA

– 3’-UAC-5’ triplet on tRNAfmet recognizes the AUG
  triplet (the start signal) when it occurs at the
  beginning of the mRNA sequence that directs
  polypeptide synthesis

– 3’-UAC-5’ triplet on tRNAmet recognizes the AUG
  triplet when it is found in an internal position in
  the mRNA sequence
How does the ribosome know where
       to start translating

– Start signal is preceded by a Shine-Dalgarno
  purine-rich leader segment, 5’-GGAGGU-3’

– Lies about 10 nucleotides upstream of the AUG
  start signal and acts as a ribosomal binding site
               Chain Elongation
• Uses three binding sites for tRNA present on the 50S
  subunit of the 70S ribosome: P (peptidyl) site, A
  (aminoacyl) site, E (exit) site.
• Requires
   – 70S ribosome
   – codons of mRNA
   – aminoacyl-tRNAs
   – elongation factors EF-Tu (Elongation factor temperature-
     unstable), EF-Ts (Elongation factor temperature-stable),
     and EF-G (Elongation factor-GTP)
   – GTP, and Mg2+
Chain Elongation
    Why is EF-Tu so important in
               E.coli?
• Involved in translation fidelity
• tRNA and aminoacid are mismatched then EF-
  Tu will not bind to t-RNA and will not deliver it
  to ribosome
• Binds activated tRNA too well and does not
  release it from ribosome
                    Elongation Steps
• Step 1
    – an aminoacyl-tRNA is bound to the A site
    – the P site is already occupied
    – 2nd amino acid bound to 70S initiation complex. Defined by the mRNA
•   Step 2
    – EF-Tu is released in a reaction requiring EF-Ts
• Step 3
    – the peptide bond is formed, the P site is uncharged
• Step 4
    –   the uncharged tRNA is released
    –   the peptidyl-tRNA is translocated to the P site
    –   EF-G and GTP are required
    –   the next aminoacyl-tRNA occupies the empty A site
            Chain Termination
• Chain termination requires
  – stop codons (UAA, UAG, or UGA) of mRNA
  – RF-1 (Release factor-1) which binds to UAA and UAG
    or RF-2 (Release factor-2) which binds to UAA and
    UGA
  – RF-3 which does not bind to any termination codon,
    but facilitates the binding of RF-1 and RF-2
  – GTP which is bound to RF-3
• The entire complex dissociates setting free the
  completed polypeptide, the release factors, tRNA,
  mRNA, and the 30S and 50S ribosomal subunits
Chain Termination
Components of Protein Synthesis
            Protein Synthesis
– In prokaryotes, translation begins very soon after
  mRNA transcription
– It is possible to have several molecules of RNA
  polymerase bound to a single DNA gene, each in a
  different stage of transcription
– It is also possible to have several ribosomes bound to
  a single mRNA, each in a different stage of translation
– Polysome: mRNA bound to several ribosomes
– Coupled translation: the process in which a
  prokaryotic gene is being simultaneously transcribed
  and translated
  Simultaneous Protein Synthesis on
            Polysomes
• A single mRNA molecule is translated by
  several ribosomes simultaneously

• Each ribosome produces a copy of the
  polypeptide chain specified by the mRNA

• When protein has been completed, the
  ribosome dissociates into subunits that are
  used again in protein synthesis
Simultaneous Protein Synthesis on
          Polysomes
               Eukaryotic Translation
• Chain Initiation:
  • the most different from process in prokaryotes
  • 13 more initiation factors are given the designation eIF
  (eukaryotic initiation factor) (Table 12.4)
          Eukaryotic Translation
• Chain elongation
  – uses the same mechanism of peptidyl transferase and
    ribosome translocation as prokaryotes
  – there is no E site on eukaryotic ribosomes, only A and
    P sites
  – there are two elongation factors, eEF-1 and eEF-2
  – eEF2 is the counterpart to EF-G, which causes
    translocation

• Chain termination
  – stop codons are the same: UAG, UAA, and UGA
  – only one release factor that binds to all three stop
    codons
    Posttranslational Modification
• Newly synthesized polypeptides are frequently modified
  before they reach their final form where they exhibit
  biological activity
   – N-formylmethionine in prokaryotes is cleaved
   – leader sequences are removed by specific proteases of the
     endoplasmic reticulum; the Golgi apparatus then directs the
     finished protein to its final destination
   – factors such as heme groups may be attached
   – disulfide bonds may be formed
   – amino acids may be modified, as for example, conversion of
     proline to hydroxyproline
   – other covalent modifications; e.g., addition of carbohydrates
Examples of Posttranslational
       Modification
          Protein Degradation


• Degradative pathways are restricted to
  – subcellular organelles such as lysosomes
  – macromolecular structures called proteosomes
Protein Degradation
          • In eukaryotes,
            ubiquitinylation
            (becoming bonded to
            ubiquitin) targets a
            protein for destruction
             – those with an N-terminus
               of Met, Ser, Ala, Thr, Val,
               Gly, and Cys are resistant
             – those with an N-terminus
               of Arg, Lys, His, Phe, Tyr,
               Trp, Leu, Asn, Gln, Asp, Glu
               have short half-lives
Acidic N-termini Induced Protein
          Degradation
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