Document Sample
Translation Powered By Docstoc
                Function of 5′ CAP

   Protect mRNA from degradation-RNAses cannot
    cleave triphosphate linkages

   Enhance the translatability of mRNAs-cap needed
    for binding of cap binding protein which is needed
    for attachment to ribosome

   Enhance the transport of the mRNA from nucleus
    to cytoplasm

   Enhance the splicing of the mRNAs
          Function of Poly (A) tail
   Protection of mRNA

   Translatability of mRNA- binding of poly (A)-binding
    protein I to the poly(A) tail region boosts the efficiency
    with which DNA is translated This protein in turn bind to
    a translation initiation factor which binds to the cap
    binding protein attached to the cap, effectively linking
    the 5’ end of the molecule to its 3’ end. This looped
    mRNA is more stable and readily translated.

   Efficient transport from the nucleus to cytoplasm
   Here, a multi-protein complex cleaves the 3'-most part of a
    newly produced RNA and polyadenylates the end produced by
    this cleavage.
   The cleavage is catalysed by the enzyme Cleavage and
    polyadenylation specificity factor (CPSF) and occurs 10–30
    nucleotides downstream of its binding site.

   This site is often the sequence AAUAAA on the RNA, but
    variants of it exist that bind more weakly to CPSF.

   Two other proteins add specificity to the binding to an RNA:
    CstF and CFI. CstF binds to a GU-rich region further
    downstream of CPSF's site.CFI recognises a third site on the
    RNA (a set of UGUAA sequences in mammals) and can recruit
    CPSF even if the AAUAAA sequence is missing.

   The polyadenylation signal – the sequence motif recognised by
    the RNA cleavage complex – varies between groups of
   The RNA is cleaved right after transcription

   When the RNA is cleaved, polyadenylation starts,
    catalysed by polyadenylate polymerase.

   Polyadenylate polymerase builds the poly(A) tail by
    adding adenosine monophosphate units from adenosine
    triphosphate to the RNA, cleaving off pyrophosphate.

   Another protein, PAB2, binds to the new, short poly(A)
    tail and increases the affinity of polyadenylate
    polymerase for the RNA.

   When the poly(A) tail is approximately 250 nucleotides
    long the enzyme can no longer bind to CPSF and
    polyadenylation stops, thus determining the length of
    the poly(A) tail
   Translation ensures that:

       polypeptide bonds are formed between adjacent
        amino acid

       that the amino acids are linked in the correct
        sequence specified by the codons in mRNA.

   mRNAs are read in the 5’→3’ direction.

   Proteins are made in the amino to carboxyl direction,
    hence the amino terminal amino acid is added first.
   The genetic code is a set of three –base code
    words, called codons, in mRNA that instruct the
    ribosome to incorporate specific amino acids into

   Each base is part of only one codon.

   There are 64 codons in all.

   Three are stop signals and the rest code for amino
   Since there are only 20 amino acids then the code is
    degenerate. This is made possible by:

       Isoaccepting tRNAs that bind the same amino acid
        although they have different anticodons.

       The third base in a codon is allowed to move slightly
        from its normal position to form a non- Watson-Crick
        pair with the anticodon.

            This allows the same aminoacyl-tRNA to pair with more than
             one codon.

            This is called wobble.
Is the Genetic code Universal?

   The genetic code is not strictly universal
    (same codons encoding the same
    information in all species).

       Termination codons in the standard genetic
        code can code for aa like tryptophan and
        glutamine in some species.
   Two events occur before protein synthesis:

       Aminacyl-tRNA synthetases join amino acids to their
        respective tRNAs: this is done in 2 steps

            First activation of the amino acid with AMP.

            Secondly tRNAs picks up the activated amino
             acids. The resulting complexes called aminoacyl-
             tRNAs are able to bind to the mRNA coding
             sequences so as to align the amino acid in the
             correct order to form the polypeptide chain

       Second ribosomes dissociate into subunits (happens
        at the end of each translation event).
Diagrammatic representation of
       tRNA molecule
             Properties of tRNA molecule:

   An amino acid is attached to transfer RNA before
    becoming incorporated into a polypeptide.
   tRNAs are responsible for aligning the amino
    acids in the correct sequence.

   Each kind of tRNA binds to a specific aa.

   It must have an anticodon, a specific
    complementary binding sequence for the correct
    mRNA codon.

   It must be recognized by a specific aminoacyl-
    tRNA synthase that adds the correct aa.
   It must have a region for the attachment of the
    specific aa specified by the anticodon.

   It must be recognized by ribosomes.

   The tRNAs are polynucleotide chains 70 to 80
    nucleotides long, each with several unique bases

   Complementary base pairing within each tRNA
    molecules causes it to be doubled back and

   Three or more loops of unpaired nucleotides are
   The aa binding site is at the 3’ end of the molecule.

   The carboxyl group of the aa is bound to the
    exposed 3’ hydroxyl group of the sugar of the
    terminal nucleotide.

   This leaves the amino group of the aa free to
    participate in peptide bonds.

   The pattern of folding results in constant distance
    between the anticodon and the aa, allowing for
    precise positioning of the aa during translation.
   Made up of 65% ribosomal RNA and 35% ribosomal proteins
    arranged into small and large subunits.

   Ribosomes consist of two subunits that fit together and work
    as one to translate the mRNA into a polypeptide chain during
    protein synthesis.

   The active part of the ribosome is RNA

Eukaryotes Ribosomes:

       Eukaryotes have 80S ribosomes, each consisting of a small (40S)
        and large (60S) subunit.

       Their large subunit is composed of a 5S RNA (120 nucleotides), a
        28S RNA (4700 nucleotides), a 5.8S subunit (160 nucleotides) and
        ~49 proteins.

       The small subunit has a 1900 nucleotide (18S) RNA and ~33
   The S number given each type of rRNA reflects the rate at
    which the molecules sediment in the ultracentrifuge. The
    larger the number, the larger the molecule (but not

   The 28S, 18S, and 5.8S molecules are produced by
    the processing of a single primary transcript from a
    cluster of identical copies of a single gene. The 5S
    molecules are produced from a different cluster of
    identical genes.
cluster of identical copies of a
          single gene
Prokaryotes Ribosomes:

   Prokaryotes have 70S ribosomes, each
    consisting of a small (30S) and a large (50S)

   Their large subunit is composed of a 5S RNA
    subunit (consisting of 120 nucleotides), a 23S
    RNA subunit (2900 nucleotides) and 34 proteins.

   The 30S subunit has a 1540 nucleotide RNA
    subunit (16S) bound to 21 proteins
      Translation in Prokaryotes

   Translation is divided into three stages:

    Initiation
   Elongation
   Termination.
   The initiation codon in prokaryotes is usually AUG.
    The initiating aminoacyl-tRNA is N-formyl-

   N-formyl-methionine is the first amino acid
    incorporated into the polypeptide chain.

   A 30S initiation complex is formed from a free 30S
    subunit plus a mRNA and fMet-tRNAfmet.

   The 16S rRNA of the 30S initiation complex first
    base pairs with a sequence called the Shine-
    Delgarno sequence upstream from the initiation
   This binding is mediated by IF3 with the
    help of IF2 and IF1.

• The initiation complex then slides along the
  mRNA until it reaches the initiation codon.

• A 30S initiation complex is formed from a
  free 30S subunit plus a mRNA and fMet-
  tRNAfmet, GTP, IF1, IF2 and IF3.
   GTP is hydrolysed after the 50S subunit
    joins the 30S complex to form the 70S
    initiation complex.

   Is the addition of other aa to the growing
    polypeptide chain.

   Takes place in three steps:

    1. EF-Tu with the help of GTP, binds an aminoacyl-tRNA to
      the A site by specific base-pairing of its anticodon and
      the complementary mRNA codon.

            The amino group of the aa at the A site is aligned with the
             carboxyl group of the preceding aa at the P site.

    2.   Peptidyl transferase forms a peptide bond between the
         peptide in the P site and the newly arrived aminoacyl-
         tRNA in the A site.
     In this process the aa that is attached to the P site is
      released from its tRNA and becomes attached to the
      aminoacyl-tRNA at the A site

     Peptidyle transferase activity resides on the 50S
      subunit (on its 23S rRNA)

3. EF-G, with GTP translocates the growing peptidyl-
  tRNA, with its mRNA codon to the P site. leaving
  the A site free for the next aminoacyl-tRNA.

     Each translocation moves the mRNA one codon’s length
      through the ribosome so that the mRNA codon
      specifying the next aa in the polypeptide chain
      becomes positioned in the unoccupied A site.
   The end of the mRNA which is transcribed
    first is also the first to be translated.

   An average sized protein of about 360 aa
    can be assembled by a prokaryote in 18
    seconds and by a eukaryotic cell in little
    over a minute.
        Elongation in eukaryotes
Elongation in eukaryotes is carried out with two
elongation factors: eEF-1 and eEF-2:

   The first is eEF-1, whose α subunit act as
    counterparts to EF-Tu.

   The second is eEF-2, the counterpart to
    prokaryotic EF-G
       Animation of translation in

         Accuracy of elongation
Mediated in two ways:

   The protein-synthesizing machinery gets rid of
    ternary complexes bearing the wrong aminoacyl-
    tRNA before GTP hydrolysis.

   It also eliminates incorrect aminoacyl-tRNA in
    the proofreading step before its amino acid gets
    incorporated into the polypeptide.

   The synthesis of the polypeptide chain is terminated by
    release factors that recognize the termination or stop codon
    at the end of the coding sequence.

   Prokaryotic translation termination is mediated by three
    factors: RF1, RF2 and RF3. RF1 recognizes UAA and UGA.
    RF3 is a GTP-binding protein that facilitates binding of RF1
    and RF2 to the ribosome.

   The release factors release the newly formed protein, the
    mRNA, and the last tRNA used,

   The ribosome dissociates into its two subunits, which are
    then reused.
                     Initiation in Eukaryotes
   Eukaryotic 40S ribosomal subunits and initiator tRNA (tRNAimet)
    locate the appropriate start codon by binding to the 5′-cap of
    an mRNA and scan downstream until they locate the first AUG.

   The initiation factors are also different than those in prokaryotic

       eIF2 is involved in binding Met-tRNAimet to the ribosome.

            eIF2 is a GTP-binding protein responsible for bringing the initiator
             tRNA to the P-site of the pre-initiation complex.

            It has specificity for the methionine-charged initiator tRNA, which is
             distinct from other methionine-charged tRNAs specific for elongation
             of the polypeptide chain.

            Once it has placed the initiator tRNA on the AUG start codon in the
             P-site, it hydrolyzes GTP into GDP, and dissociates.
   eIF2B activates eIF2 by replacing its GDP with

    eIF3 binds to the 40S ribosomal subunit and
    inhibits its reassociation with 60S subunit:

        eIF1, eIF1A, and eIF3, all bind to the ribosome
         subunit-mRNA complex.

        They have been implicated in preventing the large
         ribosomal subunit from binding the small subunit
         before it is ready to commence elongation.
   eIF4 is a cap-binding protein composed of 3

      eIF4E   has cap-binding activity

      eIFA has RNA helicase activity and unwinds
      5’-leaders of eukaryotic mRNAs.

      eIF4G  is an adaptor protein that bind to
      proteins like: eIF3 (the 40S ribosomal
      subunit binding protein) and Pab1p (a poly
      (A) tail binding protein) thereby associating
      40S subunit with both ends of mRNA and
      stimulate initiation.
   eIF5 encourages association between the 43S
    complex (comprising the 40S subunit plus
    Met-tRNAimet) and large ribosomal subunit:

        eIF5A is a GTPase-activating protein, which helps
         the large ribosomal subunit associate with the small
         subunit. It is required for GTP-hydrolysis by eIF2
         and contains the unusual amino acid hypusine.

        eIF5B is a GTPase, and is involved in assembly of
         the full ribosome (which requires GTP hydrolysis).

   eIF6 binds to the 60S subunit and blocks its
    reassociation with 40S subunit.
        Termination in Eukaryotes

   Eukaryotes have 2 release factors:

       eRF1 that recognizes all three termination

       eRF3, a ribosome-dependent GTPase that
        helps eRF1 release the finished polypeptide.
 A single mRNA molecule usually has many
  ribosomes traveling along it, in various
  stages of synthesizing the polypeptide.
  This complex is called a polysome
   Weaver R. F. Molecular Biology. Fourth edition. McGraw Hill Higher Education.


Shared By: