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Evidence for a triplet code

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					   Evidence for a triplet code
• numerical argument: to encode 20
  amino acids, a code word of at least 3 is
  necessary (41= 4; 42 = 16; 43 = 64)
• Intragenic suppressor mutations in T4
  rII locus indicated a triplet code...
  Proflavin dyes: molecules have
  the dimensions of a purine-
  pyrimidine base pair….


Distortion in helix will cause
errors during DNA
replication; leads to add or
loss of a base pair...       Frameshift
                             mutations...
Intragenic suppressor mutations…
• suppose one mutation added a base… incorrect frame leads
to mutant
• suppose a second mutation removed a base… reading frame
restored…
• if sites are close together, phenotype may be near normal
                       Sequence back in + type frame...
     (+)                                         (+)       (-)




   Wild type                                      mutant
Intragenic suppressor mutations…
• the existence of separate sites could be shown by
recombinational analysis with wildtype T4


        (+)                                 (+)       (-)




                       recombination



    Wild type                                mutant
 Take mutants that were in the (+) category and
 score for wild type phenotype in recombination
 experiments….

CAT CAT CAT CAT CAT CAT CAT CAT CAT...
CAT CAT GCA TCA TCA TCA TCA TCA TCA...

CAT CAT GCA TCA GTC ATC ATC ATC ATC...

CAT CAT GCA TCA GTC ATC GAT CAT CAT... “revertant
                                           wild type”
                                           phenotype
                       Three added bases restores
                       the correct reading frame;
                       message in phase; so “code
                       word” consists of 3 units...
C. Yanofsky: colinearity of mutations in genetic map
with amino acid changes in mutant proteins...




              Individual point mutations only change a
              single amino acid… evidence for non-
              overlapping code
   Translation in Prokaryotic
           Systems
• Three major components necessary to
  translate information in mRNA molecule
  – mRNA
  – ribosome
  – charged tRNAs
General        prokaryotic
Structure of
Ribosome




               eukaryotic
tRNA: the adaptor molecule
for protein synthesis:
mRNA  protein
General Structure of tRNA molecules

• small; approximately 75
nucleotides
• extensive intrastrand
hydrogen bonding
• “cloverleaf” structure
contains 3 characteristic
loops; anticodon loop is
responsible for pairing with
mRNA
• 3’ end of molecule
associates with specific
amino acid
In 3 dimensions, tRNAs
are L shaped...




                         Becomes
                         “charged” with
                         appropriate amino
                         acid


    Associates with
    mRNA
    How does tRNA become charged with an amino acid?

Aminoacyl tRNA
synthetases charge
tRNAs in a two-step
reaction...



  Reaction must be
  specific…different
  tRNA synthetases
  must charge the
  appropriate tRNA
  with the appropriate
  amino acid…
The two-step reaction allows for some
tRNA synthetases to “proof-read”...




                    Similar in
                    shape….              Error in
                                         charging

                                 Intermediate
                                 broken down and
                                 error corrected...
    Translation: production of a protein from mRNA
Initiation requires the
assembly of the mRNA
on the ribosome at the
                                 Small subunit first
appropriate starting             associates with
point for translation            mRNA


                     A specialized
                     charged tRNA
                     associates with
                     the small
                     subunit
                                                       P   A

       The large subunit is recruited to
       finish the complex; first tRNA is
       present in “P” site of ribosome
How is correct initiation sequence determined for
prokaryotic mRNAs?         Shine-Dalgarno sequence
                           in mRNA...




                              Start codon
Shine-Dalgarno sequence
in the mRNA is able to
base-pair with 3’ end of
RNA in small ribosomal
subunit
Elongation: assembly
of a polypeptide on the
ribosome
• charged tRNA enters
the A site via pairing to
codon
• peptidyl transferase
activity on large subunit
forms peptide bond,
transferring growing
peptide to A site

• translocation of mRNA relative
to ribosome moves nascent
polypeptide to P site
• A site now free…repeat…
Termination: mediated by
a protein release factor
• stop codon (UAG, UGA,
UAA enters “A” site)
• release factor interacts with
stop codon
• polypeptide chain is
released and ribosome
dissociates
      “Cracking the Code”
• S. Ochoa: use of the enzyme
  polynucleotide phosphorylase (PNP) to
  make synthetic RNAs
• With PNP, composition of RNA is
  random and synthesis will depend on
  amount and type of nucleotides added
  to reaction
  – e.g. all U’s will make UUUUUUUUUU…
  – all G’s will make GGGGGGGGG...
       “Cracking the Code”
• Translation of synthetic RNA could be
  accomplished in a test tube using
  extracts made from E. coli cells: e.g.:
  – Make E. coli extracts from cells grown in
    the presence of one (out of 20) “hot”
    amino acid
     • these lysates will contain ribosomes and
       charged tRNAs, one of which will be
       radioactive
  – add synthetic “mRNA”
  – test to see if radioactive amino acid is
    incorporated into proteins
      “Cracking the Code”
• UUUUU….= Phe-Phe-Phe...
• GGGGG….= Gly-Gly-Gly…etc
• Make synthetic RNAs using
  combinations of nucleotides: e.g. 3/4 U,
  1/4 G:
  – GUUUUGGUUGUU... (random polymer)
  – 8 possible codons: UUU,UUG, UGU,GUU,
    UGG, GGU, GUG, GGG
          “Cracking the Code”
• The frequency of the 8 codons will
  depend on the ratios of U and G in the
  synthetic mRNA:
  –   UUU = 3/4 X 3/4 X 3/4 = 27/64 = 42%
  –   UUG = 3/4 X 3/4 X 1/4 = 9/64 = 14%
  –   UGU = 3/4 X 1/4 X 3/4 = 9/64 = 14%
  –   GUU = 1/4 X 3/4 X 3/4 = 9/64 = 14%
  –   UGG = 3/4 X 1/4 X 1/4 = 3/64 = 5%
  –   GGU = 1/4 X 1/4 X 3/4 = 3/64 = 5%
  –   GUG = 1/4 X 3/4 X 1/4 = 3/64 = 5%
  –   GGG = 1/4 X 1/4 X 1/4 = 1/64 = 1%
          “Cracking the Code”
• Translate the synthetic mRNA in an in
  vitro protein synthesizing system. The
  frequency of the amino acids made
  should reflect the codon frequency:
  –   phe (UUU) = 42%
  –   leu (UUG) = 14%
  –   val (GUU & GUG) = 19%
  –   cys (UGU) = 14%
  –   trp (UGG) = 5%
  –   gly (GGG & GGU) = 6%
        “Cracking the Code”
• Khorana: Developed a way to
  chemically synthesize short stretches of
  ribonucleotides in an ordered array: e.g.
  – (UG)n = UGUGUGUGUGUG…
  – could give rise to two different alternating
    codons UGU GUG UGU...
  – led to synthesis of alternating polypeptide
    cys-val-cys-val...
       “Cracking the Code”
• Nirenberg: Ribosome binding assay:
  under in vitro conditions, the two
  subunits of a ribosome and the
  appropriate tRNA can assemble on a
  short RNA consisting of just a single
  codon triplet.
  – Synthesized all triplet combinations.
  – Tested to see which charged tRNA (out of
    the twenty possible) could lead to the
    assembly of a ribosome complex.
Binding of      labeled amino acid
correct
charged
tRNA will
promote
ribosome
assembly
and trap
radioactivity
on filter….
If the
incorrect       labeled amino acid
anticodon
was used in
the assembly
reaction,
then no
complex
would form
and no
radioactivity
would be
trapped on
filter...
       Features of the Code
• The code is “degenerate”: i.e. different
  codons can specify the same amino
  acid
• A substitution at the third codon position
  often encodes the same amino acid
• amino acids related in their properties
  have similarities in their codon structure;
  e.g.:
  – acidic residues are GAN (N = G, C, U or A)
  – all codons with a U in the second position
    are hydrophobic
         Codon “Wobble”
• The number of distinct tRNAs can be
  less than the number of possible
  codons.
• Because of relaxed base pairing
  interactions occurring at the third
  position of the codon and the 1st
  position in tRNA anticodon, Some
  tRNAs can recognize more than one
  codon
Codon Wobble

     3’ Val



          5’
               “Wobble” base
        CAI
        GUU
5’                   3’
        GUC
        GUA
     The Code is Universal
           (Almost)
• Cytoplasmic organelles (mitochondria
  and chloroplasts) contain their own
  genomes
• These molecules are usually present as
  a closed circular DNA molecule
• These genomes encode their own rRNA
  and tRNAs and have their own
  translation apparatus
     The Code is Universal
           (Almost)
• organelle genomes encode certain
  proteins needed for oxidative
  phosphorylation (mitochondria) and
  photosynthesis (chloroplasts)
• The genetic code used by these
  organelles is slightly different from the
  regular code:
  – human mitochondria UGA = trp, not stop
  – human mitochondria AUA = met not ile
Origin of Organelle Genomes
and Non-Nuclear Inheritance
• endosymbiont theory: organelles
  represent remnants of free-living
  organism that established symbiotic
  relationship with proto-eukaryotes
• inheritance of organelle genetic
  information is often uniparental, usually
  maternal
• Mechanism of inheritance will therefore
  be non-Mendelian, often only through
  egg cytoplasm (e.g. MERRF)

				
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posted:8/8/2011
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