Introduction
- Polynucleotide phosphorylase was first discovered from extracts of Azotobacter agile by Grunberg-Manago and Ochoa.
- Polynucleotide phosphorylase (PNPase) catalyzes the synthesis of long chain polyribonucleotides (RNA) in 5’ to 3’ direction from nucleotide diphosphates as precursors and reversibly catalyzes phosphorolytic cleavage of polyribonucleotides in 3’ to 5’ direction with a release of orthophosphate in presence of inorganic phosphate.
- PNPase is a bifunctional enzyme and functions in mRNA processing and degradation inside the cell.
- Structural and physiochemical studies in enzymes showed that it is formed of subunits. The arrangements of the subunits may vary from species to species which would alter their properties.
- These enzymes can catalyze not only the synthesis of RNA from the mixtures of naturally occurring ribonucleoside diphosphates, but also that of non-naturally occurring polyribonucleotides
Mechanism of action of Polynucleotide phosphorylase
polynucleotide phosphorylase is a bifunctional enzyme. The mechanism of action of this enzyme can be represented by following reactions:
- In E.coli, polynucleotide phosphorylase regulates mRNA processing either by adding ribonucleotides to the 3’ end or by cleaving bases in 3’ to 5’ direction. The function of PNPase depends upon inorganic phosphate (Pi) concentration inside the cell.
- The transcripts are polyadenylated using enzyme polyadenylate polymerase I (PAPI).
- After primary polyadenylylation of the transcript by PAP I, PNPase may bind to the 3′ end of the poly(A) tail. PNPase works either degradatively or biosynthetically inside the cell depending on the Pi concentration.
- Under high Pi concentration, it degrades the poly(A) tail releasing adenine diphosphates.
- If the Pi concentration is low, PAP I initiates addition of one or more nucleotides to the existing poly (A) tail and in the process generates inorganic phosphate. On dissociation of PNPase, the 3′ end again is available to PAP I for further polymerization
Functions of Polynucleotide phosphorylase
- It is involved in mRNA processing and degradation in bacteria, plants, and in
humans. - It synthesizes long, highly heteropolymeric tails in vivo as well as accounts for all of the observed residual polyadenylation in poly(A) polymerase I deficient strains.
- PNPase function as a part of RNA degradosome in E.coli cell. RNA degradosome is a multicomponent enzyme complex that includes RNaseE (endoribinuclease), polynucleotide phosphorylase (3’ to 5’ exonuclease), RhlB helicase (a DEAD box helicase) and a glycolytic enzyme enolase. This complex catalyzes 3’ to 5’ exonuclease activity in presence of ATP. In eukaryotes, the exosomes are located in nucleus and cytoplasm. Degradsomes in bacteria and exosomes in eukaryotes are associated with processing, control and turnover of RNA transcripts.
- In rDNA cloning technology, it has been used to synthesize radiolabelled polyribonucleotides from nucleoside diphosphate monomers.