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Lecture Protein Synthesis Translation Amino Acid


Lecture Protein Synthesis Translation Amino Acid

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									                   Lecture 20: Protein Synthesis: Translation

The Genetic Code is Translated on the Ribosomes

      Translation consists of 2 simultaneous actions:
           o Deciphering the genetic code
           o Formation of a protein according to the directions given by the code
      Translation takes place outside the nucleus, on the ribosome
      Two types of ribosomes:
           o Free ribosomes
           o Ribosomes attached to the endoplasmic reticulum (ER)- forms the rough
      All protein synthesis starts on free ribosomes
           o If the growing protein has a certain "signal sequence" the ribosome
              attaches to the ER (see figure, p. 313 of text)
           o Proteins made on the ER are destined to be secreted or routed to certain
              organelles such as the cell membrane or lysosomes
           o Proteins made on free ribosomes go to the cytosol, mitochondria and

Three Types of RNA are Involved in Synthesizing Proteins

      Three types of RNA (all formed in nucleus) are used in translation:
          o Messenger RNA (m-RNA): carries working copy of the code for a protein
          o Ribosomal RNA (r-RNA): furnished structure and function for ribosomes
              (60% RNA): r-RNA includes the enzyme, peptidyl transferase
          o Transfer RNA (t-RNA): carries individual amino acids from the cytosol to
              the ribosome
                   At least 20 types needed because there are 20 different types of
                      amino acids

The Ribosomes Have a Large and a Small Subunit

      Ribosomes are made in the nucleolus
      Each ribosome has a large unit and a small unit
           o Separate unless making protein
           o In eukaryotic cells subunits are designated 60S and 40S (S =
               sedimentation coefficient; large subunit has large number, sediments faster
               in ultracentrifuge)
           o Prokaryotic cells have smaller subunits: 50S and 30S
      In protein synthesis the small subunit binds to the m-RNA and then to the large
       subunit to make the complete ribosome
      Details of the ribosome assembly:
           o When the small subunit binds the m-RNA it moves along it to find the
               start codon (AUG)
                  The start codon is also used to code methionine; every eukaryotic
                   protein starts with methionine (in some cases the amino acid is
                   later removed)
         o Then a t-RNA molecule carrying methionine binds to the AUG codon
         o This causes the large subunit to bind to the complex
      Many ribosomes can attach to and read the same m-RNA
         o Produces polyribosomes

A Gene is Located in an Open Reading Frame on the m-RNA

      A gene is a sequence of codons between a start codon and a stop codon
      This structure is called an open reading frame, which means that a ribosome can
       read it without being stopped
      There can be no stop codons in the middle of a gene
      A long open reading frame will not be formed randomly very often because 3 of
       the 64 codons are stop codons (about 5%)

The Ribosome Reads the Message, One Codon at a Time

      The ribosome positions itself over the start codon and then moves along the
       messenger RNA 3 bases at a time, from codon to codon
      Movement requires energy from guanosine triphosphate (GTP)- similar to ATP
      Movement is always from the 5' direction to the 3' direction

Transfer RNA Matches Amino Acids to Codons

      Amino acids are shuttled to the ribosome by 20 types of transfer RNA (t-RNA)
      Small RNA molecules, cloverleaf structure
      Each t-RNA has a specific amino acid binding site and an anticodon site
          o Binding of amino acids requires ATP and a special enzyme (20 types of
              enzyme- one for each amino acid)
          o Anticodon site is complementary to the codon on m-RNA
          o Example: if the codon on m-RNA is CUA the t-RNA anticodon will be
          o Assures that correct amino acid is attached for each codon: the CUA/GAU
              codon/anticodon pair will cause the amino acid leucine to be added to the
Amino Acids are Added to the Protein One at a Time

      Ribosome (small subunit) has 2 binding sites for t-RNAs to bring in amino acids
          o P site has the peptide, attached to the last t-RNA
          o A site has the next amino acid attached to its t-RNA molecule
      Binding site is where t-RNA anticodon binds to codon on the m-RNA
      Steps in protein synthesis:
          o Start codon (AUG) is exposed at the P binding site and binds to the t-RNA
               for methionine
          o The next codon is exposed at the A site
          o A t-RNA with the correct anticodon binds to the A site; there are now 2
               amino acids attached to the ribosome- one at the P site and one at the A
          o The 2 amino acids are attached together, forming a peptide bond (this
               reaction is catalyzed by peptidyl transferase, an RNA enzyme); the peptide
               is attached to the A site t-RNA
          o The P site t-RNA leaves the ribosome
          o The A site t-RNA, with its peptide is transferred to the P site and the
               ribosome shifts 1 codon along the m-RNA
          o This exposes the next codon at the A site
          o The process of adding amino acids is repeated over and over until the stop
               codon is reached

                                   Initial steps: Messenger RNA is
                                  bound to ribosome with the start
                                  codon (AUG) at the P site. A
                                  transfer RNA molecule with the
                                  amino acid methionine (M) and
                                  the anticodon UAC has bound to
                                  the exposed start codon. The
                                  codon UCA is exposed at the A

                                   A second transfer RNA
                                  molecule, with the anticodon
                                  AGU and the amino acid serine
                                  (S) has bound to the A site.

                                   A peptide bond has formed
                                  between M and S and the peptide
                                  is bound to the A site. The
                                  methionine transfer RNA leaves,
                                  and the P site is exposed.

                                   The ribosome has moved along
                                  the messenger RNA one codon,
                                  bringing the peptide to the P site.
                                  This exposes the A site and the
                                  next transfer RNA, carrying
                                  alanine (A) is about to bind.

Review of the Coding: DNA -> RNA -> Protein

                  Start      1st Codon     2nd Codon 3rd Codon
 DNA code         TAC           AGT          CGG        GCT
 m-RNA code       AUG           UCA          GCC        CGA
              UAC               AGU           CGG            GCU
 Amino Acid Methionine         Serine        Alanine       Arginine

A Mutation is an Inherited Change in the Genetic Code of a Gene

      Changing the DNA bases will change the protein code
      In many cases this will be lethal and the cell will die
           o The changes may alter the active site of the protein
           o Stop codons may be inserted so that most of the gene cannot be translated
           o The protein may still work, but may have harmful side effects
      If DNA changes are passed on to the next generation the changes are called
      Mutations occur naturally due to attack of the DNA by chemicals, radiation, etc.
      Most potential mutations are repaired, but some are not
   Natural selection eliminates harmful mutations
        o In the absence of selection mutations will build up in genes; if gene is not
            needed it will be wrecked
        o Stop codon mutations will tend to destroy genes not subject to selection
        o Example: gene necessary for the production of vitamin C has been
            wrecked in many vertebrates including humans; we now must get vitamin
            C from the diet
                 The damaged enzyme is called gulonolactone oxidase; the gene is
                    located on human chromosome 8
                 Gene is damaged in primates, guinea pig, Indian fruit bat and some
   The simplest type of mutation is a point mutation; a single DNA base is changed
    to another base
   Example of point mutation: sickle cell anemia
        o A defect in hemoglobin, the protein that carries oxygen in the blood
        o A point mutation in which a single amino acid in one of the hemoglobin
            chains has been altered
        o A glutamic acid is changed to a valine in the number 6 position of the beta
        o This causes the hemoglobin to crystallize and damage the red blood cells
                 Red cells destroyed -> anemia
                 Red cells plug capillaries -> tissue damage
        o The codons for glutamic acid are: GAA and GAG
        o Codons for valine are: GUA, GUC, GUG and GUU
        o What base changes are the most likely cause of the sickle mutation?
        o The sickle gene is high in malaria areas of the world (Africa, Southeast
            Asia) because those who are heterozygous (one A gene and one S gene)
            are partially protected against malaria
                 A = normal hemoglobin gene; S = sickle hemoglobin gene
        o Those with 2 S genes have severe anemia
        o Current treatment: hydroxyurea, turns on production of fetal hemoglobin
            which relieves symptoms
        o Possible gene therapy

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