1 / 9

POLYNUCLETIDES

BIOCHEMISTRY

mprasadnaidu
Download Presentation

POLYNUCLETIDES

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. POLYNUCLEOTIDE PHOSPHORYLASE M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

  2. 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 Polynucleotide Phosphorylase Lengthened polynucleotide

  3. 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’-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

  4. 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

  5. While cracking the Genetic code, S. Ochoaused 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 U’s will make UUUUUUUUUU… • all G’s will make GGGGGGGGG...

  6. 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

  7. UUUUU…..= Phe-Phe-Phe...etc • GGGGG….= Gly-Gly-Gly…etc • 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

  8. THANK YOU

More Related