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The Genetic Code and Transcription

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U at the 5' end can pair with A or G at the 3' of mRNA. G may pair with U or C ... U or C in 2nd spot in hydrophobic AA. G or C in 2nd spot in hydrophilic AA ... – PowerPoint PPT presentation

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Title: The Genetic Code and Transcription


1
Chapter 12
  • The Genetic Code and Transcription

2
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

3
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

4
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

5
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

6
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

7
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

8
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

9
Mixed Co-Polymer
10
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

11
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

12
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

13
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

14
  • Coding dictionary for the 64 codons
  • 61 codons for AA
  • 3 codons for termination

15
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

16
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

17
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

18
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

19
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

20
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

21
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
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