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 (ΔKHS1) that severely impaired PNPase binding to RNA. Nevertheless, a residual RNA binding activity, possibly imputable to catalytic binding, could be observed even in the ΔKHS1 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-ΔKHS1-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 Δpnp mutation at 18 °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.

The KH and S1 domains of Escherichia coli polynucleotide phosphorylase are necessary for autoregulation and growth at low temperature / M.E. Matus-Ortega, M.E. Regonesi, A. Piña-Escobedo, P. Tortora, G. Deho', J. García-Mena. - In: BIOCHIMICA ET BIOPHYSICA ACTA, N. GENE STRUCTURE AND EXPRESSION. - ISSN 0167-4781. - 1769:3(2007), pp. 194-203. [10.1016/j.bbaexp.2007.01.008]

The KH and S1 domains of Escherichia coli polynucleotide phosphorylase are necessary for autoregulation and growth at low temperature

M.E. Regonesi
Secondo
;
G. Deho'
Penultimo
;
2007

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 (ΔKHS1) that severely impaired PNPase binding to RNA. Nevertheless, a residual RNA binding activity, possibly imputable to catalytic binding, could be observed even in the ΔKHS1 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-ΔKHS1-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 Δpnp mutation at 18 °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.
Cold adaptation; PNPase; Protein aggregation; RNA-binding domains
Settore BIO/18 - Genetica
2007
Article (author)
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/29351
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