Polynucleotide phosphorylase

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POLYNUCLEOTIDE PHOSPHORYLASE

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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Polynucleotide Phosphorylase
A bacterial enzyme, Polynucleotide phosphorylase,
was the first nucleic acid Synthesizing enzyme
discovered in 1955 by Marianne Grunberg-Manago
and Severo Ochoa Arthur Kornberg discovered DNA
polymerase thereafter. Polynucleotide
phosphorylase makes random RNA-like Polymers In
vitro, it catalyzes the reaction, (NMP)n
NDP (NMP)n1 Pi
Lengthened polynucleotide
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(No Transcript)
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Activity of the Enzyme

• It is not template-dependent
• Uses 5-diphosphates of ribonucleosides as
  substrates (No 5-deoxyribonucleosides or rNTPs)
• But, RNA formed contains usual 3,5-phosphodieste
  r linkages
• As it does not use a template, the polymer it
  forms does not have a specific base sequence
• The reaction proceeds equally well with any of
  the four nucleoside diphosphates
• The resulting polymer reflects the relative
  concentration of the substrates in the medium

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• The enzyme can be used to prepare RNA polymers
  with many different base sequences and
  frequencies
• Synthetic RNA polymers of this sort were critical
  for deducing the Genetic code for the amino
  acids.
• The reaction is reversible in the direction of
  breakdown of polyribonucleotides
• Probably, the function in the cell Degradation
  of mRNAs to nucleoside diphosphates

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• While cracking the Genetic code, S. Ochoa used
  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 Us will make UUUUUUUUUU
• all Gs will make GGGGGGGGG...

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• Translation of synthetic RNA could be
  accomplished in a test tube using extracts made
  from E. coli cells
• 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

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• UUUUU.. Phe-Phe-Phe...etc
• GGGGG. Gly-Gly-Glyetc
• 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

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THANK YOU