A novel mutation in the KH domain of polynucleotide phosphorylase affects autoregulation and mRNA decay in Escherichia coli

Polynucleotide phosphorylase (PNPase) is a key 3'-5' exonuclease for mRNA d ecay in bacteria. Here, we report the isolation of a novel mutant of Escher ichia coli PNPase that affects autogenous control and mRNA decay. We show t hat the inactivation of PNPase by a transposon insertion increases the half -life of galactokinase mRNA encoded by a plasmid, When the bacteriophage la mbda int gene retroregulator (sib/tl) is placed between pgal and galK, it s everely diminishes galactokinase expression because of transcription termin ation. The expression of galK from this construct is increased by a single base mutation, sib1, which causes a partial readthrough of transcription at tl. We have used this plasmid system with sib1 to select E. coli mutants t hat depress galK expression, Genetic and molecular analysis of one such mut ant revealed that it contains a mutation in the pnp gene, which encodes the PNPase catalytic subunit or. The mutation responsible (pnp-71) has substit uted a highly conserved glycine residue in the KH domain of PNPase with asp artate. We show that this G-570D substitution causes a higher accumulation of the ol-subunit as a result of defective autoregulation, thereby increasi ng the PNPase activity in the cell. The purified mutant alpha-subunit shows the same electrophoretic mobility in denaturing gels as the wild-type subu nit, as expected. However, the mutant protein present in crude extracts dis plays an altered electrophoretic mobility in nondenaturing gels that is ind icative of a novel enzyme complex. We present a model for how the pnp-71 mu tation might affect autoregulation and mRNA decay based on the postulated r ole of the KH domain in RNA-protein and protein-protein interactions.