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Mutational analysis of polynucleotide phosphorylase from Escherichia coli
Anne Jarrige1, Dominique Bréchemier-Baey, Nathalie Mathy
1UPR 9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France.
Abstract:
Polynucleotide phosphorylase (PNPase), a homotrimeric exoribonuclease present in bacteria, is involved in mRNA degradation. In Escherichia coli, expression of this enzyme is autocontrolled at the translational level. We introduced about 30 mutations in the pnp gene by site-directed mutagenesis, most of them in phylogenetically conserved residues, and determined their effects on the three catalytic activities of PNPase, phosphorolysis, polymerisation and phosphate exchange, as well as on the efficiency of translational repression. The data are presented and discussed in the light of the crystallographic structure of PNPase from Streptomyces antibioticus. The results show that both PNPase activity and the presence of the KH and S1 RNA-binding domains are required for autocontrol. Deletions of these RNA-binding domains do not abolish any of the three catalytic activities, indicating that they are contained in a domain independent of the catalytic centre. Moreover, the catalytic centre was located around the tungsten-binding site identified by crystallography. Some mutations affect the three catalytic activities differently, an observation consistent with the presence of different subsites.
Insights
Polynucleotide phosphorylase (PNPase) in E. coli requires RNA-binding domains and catalytic activity for translational autocontrol. Mutations reveal distinct subsites within the enzyme’s catalytic center.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Polynucleotide phosphorylase (PNPase) is a crucial bacterial exoribonuclease involved in mRNA degradation.
- In Escherichia coli, PNPase expression is regulated at the translational level, a process known as autocontrol.
Purpose of the Study:
- To investigate the roles of PNPase's catalytic activities and RNA-binding domains in translational autocontrol.
- To identify the location of the catalytic center and explore potential distinct subsites.
Main Methods:
- Site-directed mutagenesis was used to introduce approximately 30 mutations into the pnp gene, targeting conserved residues.
- The effects of these mutations on PNPase's phosphorolysis, polymerization, and phosphate exchange activities were assessed.
- The impact on translational repression efficiency was also determined.
Main Results:
- Both PNPase catalytic activity and the presence of KH and S1 RNA-binding domains are essential for translational autocontrol.
- Deletion of RNA-binding domains did not affect catalytic activities, suggesting their independence from the catalytic center.
- The catalytic center was localized near the tungsten-binding site, and some mutations differentially affected catalytic activities, indicating distinct subsites.
Conclusions:
- PNPase's translational autocontrol mechanism relies on the interplay between its catalytic functions and RNA-binding capabilities.
- The catalytic center is structurally distinct from the RNA-binding domains.
- The PNPase catalytic center likely comprises multiple subsites responsible for its diverse enzymatic activities.