POLYNUCLEOTIDE

PHOSPHORYLASE
M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.
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)
n+1
+ Pi

Lengthened
polynucleotide
It is not template-dependent
Uses 5-diphosphates of ribonucleosides as substrates
(No 5-deoxyribonucleosides or rNTPs)
But, RNA formed contains usual 3,5-phosphodiester
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

Activity of the Enzyme
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
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...

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

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
THANK YOU