The Genetic Code

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The Genetic Code

• ?? 200431060029

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Do you know these people?
Robert Holley, the discoverer of the transfer RNA
- tRNA.
George Gamow, Russian physicist, founded the "RNA
Tie Club" in 1954.
arshall Nirenberg, the scientist that deciphered
the genetic code in 1961.
Har Gobind Khorana, creator of new methods to
produce synthetic nucleic acids.
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Preview

• The translation of genetic and is mediated by
  special adaptor molecules knows as transfer
  RNAs(tRNAs).
• Three consecutive nucleotides are known as
  codons.
• With four possible nucleotides at each position,
  the total number of permutations of there
  triplets is 64.

The DNA molecule, the carrier of the genetic
information.
RNA, a molecule which resembles DNA, is however
single-stranded.
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OutLine

• The Code Is Degenerate
• Three Rules Govern the Genetic Code
• Suppressor Mutations Can Reside in the Same or a
  Different Gene
• The Code is Nearly Universal

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The Code Is Degenerate(???)

1. Many amino acides are specified by more than one
   codon, the phenomenon called degeneracy.
2. When the first two nucletides are identical, the
   third nucleotide can be either cytosine or uracil
   and the codon will still code for the same amino
   acide
3. Often,adenine and guanine are similarly
   interchangeable

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The Code Is Degenerate(???)

• Not all degeneracy is based on equivalence of the
  first two nucleotides
• There can be great variation in the AT/GC ratios
  in the DNA of various organisms without
  correspondingly large changes in the relative
  proportion of amino acids in the proteins

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The Genetic Code is Unambiguous

• In general, no codon specifies more than one
  amino acid. The exceptions so far are AUG, UGA
  and UAG. In the first case, AUG specifies both
  Methionine and N-formyl-Methionine, which is used
  to initiate protein synthesis in bacteria. In the
  second case, UGA specifies the twenty-first
  amino-acid selenocysteine as well as being a stop
  codon. And, in the last case, UAG specifies the
  twenty second amino acid (the most recent to be
  added to the list), pyrrolysine.

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Perceiving Order in the Makeup of the Code

• The code evolved in such a way as to
  minimize the deleterious effects of mutations
• Mutation in the first position of a codon will
  often give a similar amino acid.
• Codons with pyrimidines in the second position
  specify mostly hydrophobic amino acids,with
  purines in the second position correspond mostly
  to polar amino acids.
• Change in the third position rarely will a
  different amino acid be specified,even
  transversion.

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Perceiving Order in the Makeup of the Code

1. Whenever the first two positions of a codon are
   both occupied by G or C, each of the four
   nucleotides in the third position specifies the
   same amino acid.
2. Whenever the first two positions of the codon are
   both occupied by A or U, the identity of the
   third nucleotide does make a difference.

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Wobble in the Anticodon

1. The base at the 5 end of the anticodon is no as
   spatically confined as the other two allowing it
   to form hydrogen bonds with any of several based
   located at the 3 end of a codon.

Base in Anticodon Base in Codon
G C A U I U or C G U A or G A,U,or C
Paring Combinations with the Wobble Concept
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Wobble in the Anticodon

• The wobble rules do not permit any single tRNA
  molecule to recognize four different codons.
• Questionwhy the wobble is in the 3 position of
  the codon?

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Wobble in the Anticodon

• The three anticodon base all point in roughly
  the same direction, with their exact
  conformations largely determined by
  stackinginteractions between the flat surfaces of
  the bases.The first (5) anticodon base is at the
  end of the stack and is perhaps less restricted
  in its movements than the other two anticodon
  bases.

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Three Codons Direct Chain Termination

• UAA,UAG,UGA are read not by special tRNA,
  but by specific proteins known as release
  factors(RF1 and RF2 in bacteria and eRF1 in
  eukaryotes). Release factors enter the A site of
  the ribosome and trigger hydrolysis of the
  peptidyl-tRNA occupying the P site, resulting in
  the release of the newlysynthesized protein.

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How the Code Was Cracked

• The first steps to solving the Genetic
• Code depended on the development of a
• cell-free in vitro translation system by Paul
• Zamecnik (right). This system which
• consisted of a membrane-free cell
• supernatent, ATP, GTP, radioactively
• labelled amino-acids and RNA, was
• capable of directing the synthesis of
• radioactively labelled protein.

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Stimulation of Amino Acid Incorporation by
Synthetic mRNAs

• The dependence of cell extracts on externally
  added mRNA provided an opportunity to elucidate
  the nature of the code using synthetic
  polyribonucleotides. These synthetic templates
  were created using the enzyme polynucleotide
  phosphorylase,which catalyzes the reaction
• XMPn XDP
  XMP n1
• Polynucleotide phosphorylase is normally
  responsible for breaking down RNA.Howere, by use
  of high nucleoside diphosphate concentrations
  this enzyme can be made to catalyze the formation
  of internucleotide 3 5 phosphodiester
  bonds and thus make RNA molecules.
• Addition of two or more different
  diphosphates produces mixed copolymers such as
  poly-AU poly-AC poly-CU and poly-AGCU.

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Stimulation of Amino Acid Incorporation by
Synthetic mRNAs
The figure shows the reversible reactions of
synthesis or degradation of polyadenylic acid
catalyzed by the enzyme polynucleotide
phosphorylase
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Poly-U Code for Polyphenylalanine

• A high magnesium concentration circumvents the
  need for initiation factors and the special
  initiator fMet-tRNAm allowing chain initiation to
  take place without the proper signals in the
  mRNA.
• Poly-U selects phenylalanyl tRNA molecules
  exclusively , thereby forming a polypeptide chain
  containing only pheny-lalanine.
• On the basis of analogous experiments with
  poly-C and poly-A,CCC was assigned as a proline
  codon and AAA as a lysine codon.
• The guanine residues in poly-G firmly hydrogen
  bond to each other and form multistranded triple
  helicase that do not bind to ribosomes.

The experiment which used uracil (U) as a
template produced a protein entirely made up of
the amino acid phenylalanine (F). The first
letter of the genetic code was hence
identified.
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Mixed Copolymers Allowed Additional Codon
Assignments

• This use of homopolymers is clearly quite
  limited. The use of random mixed copolymers
  helped to extend the utility of the system and
  the information obtained from it.
• Random copolymers can be synthesized from a
  mixture of two ribonucleotides with
  polynucleotide phosphorylase. Thus if ADP and CDP
  are used in a 51 ratio, then the frequency of
  each possible triplet in the synthesized RNA will
  vary according to this ratio. For example, AAA
  triplets will be found 100 times more frequently
  than CCC triplets.

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Transfer DNA Binding to Defined Trinucleotide
Codons

• aminoacylated tRNAs could be bound to ribosomes
  if the ribosomes contained trinucleotides acting
  as mRNA.

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Codon Assignments from Repeating Copolymers

• tri- and tetra-nucleotides could be polymerized
  into polymers with repeating sequences that could
  be used in cell-free in vitro translation assays
  .
• In the case of trinucleotides, three polypeptides
  will be synthesized, each of which is a
  homopolymer of a single amino acid.

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Three Rues Govern The Genetic Code

1. Codons are read in a 5 to 3 direction.
2. Codons are nonoverlapping and the message
   contains no gaps.
3. The message is translated in a fixed reading
   frame, which is set by the initiation codon.

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Three Kinds of Point Mutations Alter the Genetic
Code

• missense mutationan alteration that changes a
  condon specific for one amino acid to a codon
  specific for another amino acid .
• nonsense/stop mutation an alteration causing a
  change to a chain-termination codon.
• Frameshift mutation insertions or deletions of
  one or a smal number of base pairs that alter the
  reading frame.

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Genetic Proof that the Code is Read in Units of
Three

• The finding indicated that the overall
  coding capacity of the gene had been
• chiefly left unaltered despite the presence of
  three mutations, each of which alone,
• or any two of which alone, would have drastically
  altered the reading frame of the
• genes message. Because the gene could tolerate
  three insertions but not one or
• two, the genetic code must be read in units of
  three.

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Suppressor Mutations can Reside in The Same or a
Different Gen
Suppressor genesgenes that cause suppression of
mutations in other genes.
One example of intragenic supression is missense
mutation.The effect can sometime be reversed
through an additional missense mutation in the
same gene Another example of intragenic
frameshift mutation.
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Suppressor Mutations can Reside in The Same or a
Different Gen

• A deletion in the nucleotide coding sequence can
  result in an incomplete, inactive poly peptide
  chain.
• (b) The effect of the deletion, shown in panel a,
  can be overcome by a second mutation, an
  insertion in the coding sequence. This insertion
  results in the production of a complete
  polypeptide chain having two amino acid
  replacements. Depending on the change in
  sequence, the protein may have partial or full
  activity.

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Intergenic Suppression Involves Mutant tRNAs

• Suppressor genes do not act by changing the
  nucleotide sequence of a mutant gen.Instead, they
  change the way the mRNA template is read.
• nonsense mutations A mutation in the anticodon
  of tRNA that alters the anticodon so it is now
  complementary to a nonsense codon allowing the
  tRNA to insert its cognate amino acid at this
  nonsense codon during translation.
• If a mutation occurs in the DNA that changes
  the AAG codon in the mRNA to UAG, the UAG codon
  will be read as a stop signal and the translation
  product will be a truncated (short) usually
  nonfunctional polypeptide.

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Nonsense Suppressors also Read Normal Termination
Signals

• The act of nonsense suppression can be viewed as
  a competition between the suppressor tRNA and the
  release factor.
• When a stop codon comes into the ribosomal A site
  , either read-through or polypeptidechain
  termination will occur, depending on which
  arrives first.

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Proving the Vality of the Genetic Code

• A classic and instructive experiment in 1966
  helped to validate the genetic code.

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The Code is Nearly Universal

• The results of large-scale sequencing of
  genomes have confirmed the universality of the
  genetic code.

Benefits of the universal codes 1. Allow us to
directly compare the protein coding sequences
among all organisms. 2. Make it possible to
express cloned copies of genes encoding useful
protein in different host organism. Example
Human insulin ecpression in bacteria)
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The Code is Nearly Universal

• However, in certain subcellular organelles,
  the genetic code is slightly different from the
  standard code.
• Mitochondrial tRNAs are unusual in the way
  that they decode mitochondrial messages.
• Only 22 tRNAs are present in mammalian
  mitochondria. The U in the 5 wobble position of
  a tRNA is capable of recognizing all four bases
  in the 3 of the codon.

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The Code is Nearly Universal
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The Code is Nearly Universal