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 decay in bacteria. Here, we report the isolation of a novel mutant of Escherichia coli PNPase that affects autogenous control and mRNA decay. We show that the inactivation of PNPase by a transposon insertion increases the half-life of galactokinase mRNA encoded by a plasmid. When the bacteriophage lambda int gene retroregulator (sib/tI ) is placed between pgal and galK, it severely diminishes galactokinase expression because of transcription termination. The expression of galK from this construct is increased by a single base mutation, sib1, which causes a partial readthrough of transcription at tI. We have used this plasmid system with sib1 to select E. coli mutants that depress galK expression. Genetic and molecular analysis of one such mutant revealed that it contains a mutation in the pnp gene, which encodes the PNPase catalytic subunit alpha. The mutation responsible (pnp-71 ) has substituted a highly conserved glycine residue in the KH domain of PNPase with aspartate. We show that this G-570D substitution causes a higher accumulation of the alpha-subunit as a result of defective autoregulation, thereby increasing the PNPase activity in the cell. The purified mutant alpha-subunit shows the same electrophoretic mobility in denaturing gels as the wild-type subunit, as expected. However, the mutant protein present in crude extracts displays an altered electrophoretic mobility in non-denaturing gels that is indicative of a novel enzyme complex. We present a model for how the pnp-71 mutation might affect autoregulation and mRNA decay based on the postulated role of the KH domain in RNA-protein and protein-protein interactions.
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apdo Postal 14-740, México DF 07000, México.
J
J
García-Mena
A
A
Das
A
A
Sánchez-Trujillo
C
C
Portier
C
C
Montañez
D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005686:Galactokinase / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006706:Homeostasis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013329:Structure-Activity Relationship
D012329:RNA, Bacterial; D012333:RNA, Messenger; D005686:Galactokinase; D011117:Polyribonucleotide Nucleotidyltransferase; D005998:Glycine