The Genetic Code and Transcription

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Chapter 12

• The Genetic Code and Transcription

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DNA to Protein

• DNA sequence ultimately dictates the end products
  of most proteins end product of most genes
• DNA is transferred to RNA transcription
• RNA acts as a message for ribosomes to make
  protein translation

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Genetic Code Characteristics

• Genetic code is linear ribonucleotides act as
  letters complementary to bases in DNA
• Word is 3 letters long, all but 3 combos or
  codons, specify an amino acid triplet codon
• Code is unambiguous each codon specifies a
  single AA
• Code is degenerate a given AA may have more
  than one codon (not Met or Trp)
• Code has 1 start or initiator and 3 stop or
  termination codons signals for translation
• No punctuation, commaless read one codon right
  after the other
• Code is non-overlapping 12 nucleotide is part
  of only 1 codon
• Code is universal very few exceptions in codon
  usage all organisms use the same code for the
  most part

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Early Studies

• Thought that DNA transferred to RNA of ribosome
  for protein synthesis
• Evidence became available of an unstable
  intermediate but RNA of ribosome was very stable
  found mRNA
• tried to determine how 4 letters could encode 20
  AA
• theoretically it must be 3 letters per AA

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Cricks Predictions

• Studying frameshift mutations in phage T4
• Insert 1 or 2 nucleotides results in an out of
  frame mutation but 3 nucleotides put things back
  in frame

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Deciphering the Code

• Used 2 different types of experiments
• in vitro (cell-free) protein synthesizing system
• found enzyme polynucleotide phosphorylase make
  polypeptide on mRNA in cell free system

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Cell-Free Polypeptide Synthesis

• Use polynucleotide phosphorylase to make an
  artificial mRNA can determine the sequence
  based on the concentration of the ribonucleotide
• In vitro mixture of ribosomes, tRNA, amino acids
  and other molecules essential to translation
• add the mRNA to serve as the template

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Homopolymers and Co-Polymers

• Homopolymers are made of one nt and translated
• UUU phe AAA lys CCC pro GGG couldnt be
  determined because folding back on itself
• Mixed co-polymers mix the nt and predict
  sequence based on amount of nt present
• could not get exact sequence but could determine
  the nucleotides in the codon

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Mixed Co-Polymer
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Triplet Binding Assay

• Used to determine the order of triplets
• RNA only 3 nt long can act as triplet bind to
  the anti-codon of tRNA
• Synthesize triplet RNA in lab to be template,
  needed to figure out how to determine the tRNA-AA
  attached
• Made radioactive AA and charged the tRNA, use
  codon composition to narrow range to test with
  each triplet
• Filter thru nitrocellulose paper if the paper
  is radioactive, then it is that AA, no
  radioactivity, then AA cant bind the triplet

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Conclusions

• 26 triplets for 9 AA, eventually IDed 50 of 61
• Major conclusions
• genetic code is degenerate 1 AA with more than
  1 codon
• code is unambiguous only 1 AA per codon

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Repeating Co-Polymers

• Made short repeating mRNA UG repeats
• made 2 codons depending where the ribosome starts
  UGU or GUG
• Also made 3, 4 etc repeats
• Made assignments for all codons and AA

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Example

• UUCUUC trinucleotide that has 3 possible ORF
  UUC, UCU, CUU got peptides with phe, ser and
  leu but not sure which codon belonged to AA
• Made dinucleotide UCUCUC. codons UCU and CUC
  got peptides leu and ser
• UCU is either leu or ser UUC or CUU encodes leu
  or ser and then the other one encodes phe
• Made tetranucleotides UUACUUAC. codons UUA,
  UAC, ACU, CUU got peptides leu, thr and tyr
• CUU must add leu or ser (from above) but no ser
  so it is codon for leu UCU is common to tri and
  di-nt so it is ser UCC becomes phe by
  elimination
• Do more of these to figure out the rest GAUA
  has at least 2 of the termination codons because
  only a few AA incorporated before stopping

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• Coding dictionary for the 64 codons
• 61 codons for AA
• 3 codons for termination

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Degeneracy

• Code is degenerate almost all AA are specified
  by 2, 3 or 4 codons
• ser, arg, and leu 6 codons each
• trp and met 1 codon each
• Pattern of degeneracy most sets that code an AA
  have 1st 2 nt in common

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Wobble Hypothesis

• 3rd position of codon is more flexible in
  base-pairing rules
• Allows single tRNA to pair with more than 1 codon
• U at the 5 end can pair with A or G at the 3 of
  mRNA
• G may pair with U or C
• Inosine may pair with C, U, or A
• Minimum of 30 different tRNA for 61 triplets
• bacteria 30-40, animals and plants 50

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Ordered Nature of Code

• Chemically similar AA share 1 or 3 middle bases
  in the triplets coding them
• U or C in 2nd spot in hydrophobic AA
• G or C in 2nd spot in hydrophilic AA
• Buffers the mutations on proteins insert
  chemically AA function of protein may not be
  altered noticeably

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Intitation

• In bacteria insert a modified met
  n-formylmethionine (f-Met) in the initial AA of
  all proteins AUG codon
• called the initiator codon
• other AUGs in the message get a regular met
• Rarely GUG can insert fmet in initiator site,
  unsure of reason
• Formyl group or entire f-met is removed from
  protein after synthesis is complete
• AUG initiator codon in eukaryotes also insert met
  but not formylated one

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Termination

• UAG, UAA and UGA are termination codons that STOP
  synthesis
• No amino acid and no tRNA recognition
• If mutation results in any of these sequences
  see termination causing an incomplete peptide
  so also called non-sense mutation

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Universal Code

• For the most part all codons encode the same
  amino acid in all organisms
• There are some rare exceptions to this in
  mitochondria and some small single cell organisms

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Overlapping Genes

• Viruses and maybe some simple organisms have
  multiple initiation spots in mRNA to make
  multiple proteins specify more than 1 peptide
• called overlapping genes
• X174 has 5386 nt so can make 1795 AA (about 2
  proteins worth), actually make 11 proteins of
  2300 AA overlapping genes