title: Mutational analysis of polynucleotide phosphorylase from Escherichia coli.
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.
authors.affiliation: UPR 9073 du CNRS, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Jarrige
authors.forename: Dominique
authors.initials: D
authors.lastname: Bréchemier-Baey
authors.forename: Nathalie
authors.initials: N
authors.lastname: Mathy
authors.forename: Ophélie
authors.initials: O
authors.lastname: Duché
authors.forename: Claude
authors.initials: C
authors.lastname: Portier
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D012329:RNA, Bacterial; D011117:Polyribonucleotide Nucleotidyltransferase