Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12.

Abstract

The isolation of a temperature-sensitive allele of RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that RNase H and polynucleotide phosphorylase (PNPase) are required for cell viability and mRNA turnover in Escherichia coli. Double-mutant strains carrying thepnp-7 and rnb-500 alleles (PNPase deficient and RNase II thermolabile) ceased growing in Luria broth within 30 min after shift to the nonpermissive temperature. Cessation of growth was accompanied by an accumulation of mRNA fragments 100-1500 nucleotides long. In contrast, single-mutant and wild-type control strains grew normally at the nonpermissive temperature and did not accumulate mRNA. No significant changes in rRNA patterns were observed in any of the strains.In vitro, both polynucleotide phosphorylase (PNPase) (8) found no difference in the in vivo rate of mRNA degradation in either the absence of RNase II activity or in the presence of a 10-fold increase over wild-type levels.In this communication, it is shown that cells lacking both PNPase and RNase II are inviable. By using the newly isolated rnb-SOO allele, which encodes thermolabile RNase II activity, it has been possible to demonstrate that the loss of cell viability that occurs in a pnp-7 rnb-SOO strain at nonpermissive temperatures is related to an in vivo accumulation of RNA species 100-1500 nucleotides long and to an increase in the chemical half-life of total mRNA. In addition, it has been shown that the inviability of such double mutants, as well as the in vivo thermolability of RNase II, is partially dependent on the growth medium.

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

“…In vivo, the final breakdown of mRNA to the mononucleotide level is dependent on two exoribonucleases, PNPase and the hydrolytic enzyme RNase II. These enzymes can substitute for each other to degrade RNA, and the presence of either one of these enzymes is required for cell viability and mRNA turnover (Donovan and Kushner, 1986). In extracts from E. coli grown at 37ЊC, the mode of RNA degradation was shown to be predominantly hydrolytic, while PNPase was responsible for only 10% of the total processive 3Ј→5Ј RNA degradation (Deutscher and Reuven, 1991).…”

Degradation of mRNA in Escherichia coli

“…RNase II uses water as a nucleophile, liberating nucleotide monophosphates, whereas PNPase uses phosphate ion as a nucleophile to generate nucleotide diphosphates (21). Nevertheless, to some degree these enzymes play a functionally interchangeable role in the cell, since E. coli mutants lacking either enzyme are viable, but mutants lacking both enzymes become inviable (22).…”

Multiple Exoribonucleases Catalyze Maturation of the 3′ Terminus of 16S Ribosomal RNA (rRNA)

“…This was necessary because double mutant strains lacking RNase II and PNPase (13) or RNase R and PNPase (14,15) are inviable, and cells lacking RNase PH and PNPase are slowed in growth (16). The mutant strain was grown at 37°C and transferred to 44°C for 1 h to inactivate PNPase such that the maturation of 16S RNA could be examined under conditions in which the activities of four exoribonucleases were lacking.…”