The KH and S1 domains of Escherichia coli polynucleotide phosphorylase are necessary for autoregulation and growth at low temperature.
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.
Matus-Ortega
Maura Epifanía
ME
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados, Apartado Postal 14-740, Mexico DF 07360 México, Mexico.
Regonesi
Maria Elena
ME
Piña-Escobedo
Alberto
A
Tortora
Paolo
P
Dehò
Gianni
G
García-Mena
Jaime
J
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
D012329:RNA, Bacterial; D012333:RNA, Messenger; D011117:Polyribonucleotide Nucleotidyltransferase