An Escherichia coli Mutant with Increased Messenger Ribonuclease Activity

Abstract

A temperature-sensitive mutant strain of Escherichia coli exhibits a remarkable increase in RNase activity when grown at its nonpermissive temperature. During growth at the nonpermissive temperature, there is an increase in the extent of breakdown of pulse-labeled RNA and a decrease in the functional lifetime of the mRNA for the lac operon. T7 RNA, which is usually stable in E. coli, is also stable in this strain at the nonpermissive temperature. It is possible that the RNase measured is part of the normal mechanism of mRNA degradation in the cell. A mechanism for mRNA degradation that requires the combined action of endonuclease(s) and 3' to 5' exonuclease(s) is proposed.An understanding of the mechanism by which mRNA is degraded could be useful for the analysis of a number of fundamental cellular processes. In order to study these mechanisms, we have been trying to isolate mutant strains modified in mRNase activity and to identify the altered enzymes. Assuming that an enzyme with mRNase activity is indispensable for normal growth, we had to resort to the isolation of conditional-lethal mutants. One procedure is to look for RNase-less strains among a collection of temperature-sensitive mutants. A disadvantage of this approach is that if a reduced level of a given RNase is not accompanied by significant alteration in mRNA lifetime, it is possible that the enzyme is present in the cell, but is unstable and destroyed during preparation of extracts. Therefore, we chose instead to look for mutants with increased RNase activity at a high temperature among a collection of temperature-sensitive mutants. It seemed possible that if the increased activity were involved in mRNA breakdown, certain readily identifiable physiological consequences could follow, e.g., a reduction in functional lifetime of mRNA.Recently we isolated a class of temperature-sensitive mutants of Escherichia coli that show an increased sensitivity to starvation, (designated sts mutants, starvation-temperature sensitive, refs. 1 and 2). These mutants are temperature sensitive (grow at 300C but not at 430C) and cannot recover from glucose starvation. Among the members of this class, we identified strains altered in the ability to synthesize proteins due to defects in ribosomal (1, 2) as well as in supernatant components (3). Since mRNA synthesis is reduced during starvation (4), it seemed reasonable to assume that strains with increased mRNase activity might have insufficient mRNA for necessary turnover processes and would fail to recover from a period of extended starvation. Therefore, we tested sonicates of 150 such sts strains in a standard assay for RNase II (5) at 300 and 430C. This assay was chosen because of its simplicity and because RNase II has already been implicated in mRNA breakdown in E. coli (6). We found eight strains with an increased RNase activity. The studies reported here were done with one of these strains, designated N4752. Our studies suggest that mRNA degradation is altered in strain N4752. RESULTS Strains, growth, and ...

A mutation affecting degradation of stable RNA inEscherichia coli

“…The participation of a pre-existing ribonuclease in a starvation degradation process is in accord with the finding of Goldberg [ 121 that preexisting protease participates in protein degradation during starvation of E. colt The teleological arguments for having preexisting enzymes participate in essential processes during starvation when metabolic activities should be kept to a minimum are obvious, and clearly degradation of one molecule to completion before starting degradation of the other could be of great selective advantage to a starving cell. The experiments reported here with strain N4752 together with those presented earlier [4,5] show that the same lesion changed mRNA as well as stable RNA degradation, and therefore they imply that a similar mechanism is responsible for degradation of all RNA species in E. coli. If degradation of stable RNA during starvation is an essential function for survival (a generally accepted, even though unproven assumption), these experiments raise the possibility that perhaps the instability of mRNA is a by-product for the necessity of providing the cell with a mechanism to degrade its stable RNA.…”

Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli.

“…N4021 was an recA56, rnc-105 strain constructed as follows. A Pl-mediated transduction (15) was carried out with JC10240 as donor and N2100 as recipient with selection for tetracycline resistance (Tetr). A resulting Rec-recombinant, N4019, was then used as a recipient in a P1-mediated transduction with D10 (16) as a donor and selection for sorbitol independence (Srl+).…”

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

“…Kivity-Vogel and Elson (6) and Lennette et al. (7) reported that the in vivo rate of mRNA degradation was proportional to the level of RNase II present in the cell. On the other hand, Donovan and Kushner (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.…”