title: The KH and S1 domains of Escherichia coli polynucleotide phosphorylase are necessary for autoregulation and growth at low temperature.
abstract: PNPase is a phosphate-dependent exonuclease of Escherichia coli required for growth in the cold. In this work we explored the effect of specific mutations in its two RNA binding domains KH and S1 on RNA binding, enzymatic activities, autoregulation and ability to grow at low temperature. We removed critical motifs that stabilize the hydrophobic core of each domain, as well as made a complete deletion of both (DeltaKHS1) that severely impaired PNPase binding to RNA. Nevertheless, a residual RNA binding activity, possibly imputable to catalytic binding, could be observed even in the DeltaKHS1 PNPase. These mutations also resulted in significant changes in the kinetic behavior of both phosphorolysis and polymerization activities of the enzyme, in particular for the double mutant Pnp-DeltaKHS1-H. Additionally, PNPases with mutations in these RNA binding domains did not autoregulate efficiently and were unable to complement the growth defect of a chromosomal Deltapnp mutation at 18 degrees C. Based on these results it appears that in E. coli the RNA binding domains of PNPase, in particular the KH domain, are vital at low temperature, when the stem-loop structures present in the target mRNAs are more stable and a machinery capable to degrade structured RNA may be essential.
authors.affiliation: Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico DF 07360 México, Mexico.
authors.forename: Maura Epifanía
authors.initials: ME
authors.lastname: Matus-Ortega
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Regonesi
authors.forename: Alberto
authors.initials: A
authors.lastname: Piña-Escobedo
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortora
authors.forename: Gianni
authors.initials: G
authors.lastname: Dehò
authors.forename: Jaime
authors.initials: J
authors.lastname: García-Mena
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006706:Homeostasis; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D011117:Polyribonucleotide Nucleotidyltransferase