RNase R is a highly unstable protein regulated by growth phase and stress - PubMed (original) (raw)
RNase R is a highly unstable protein regulated by growth phase and stress
Chenglu Chen et al. RNA. 2010 Apr.
Abstract
RNase R is an important exoribonuclease that participates in the degradation of structured RNAs in Escherichia coli. In earlier work, it was shown that RNase R levels increase dramatically under certain stress conditions, particularly during cold shock and stationary phase. However, the regulatory processes that lead to this elevation are not well understood. We show here that the increase in RNase R in stationary phase is unaffected by the global regulators, RpoS and (p)ppGpp, and that it occurs despite a major reduction in rnr message. Rather, we find that RNase R is a highly unstable protein in exponential phase, with a half-life of approximately 10 min, and that the protein is stabilized in stationary phase, leading to its relative increase. RNase R is also stabilized during cold shock and by growth in minimal medium, two other conditions that lead to its elevation. These data demonstrate that RNase R is subject to regulation by a novel, posttranslational mechanism that may have important implications for our complete understanding of RNA metabolism.
Figures
FIGURE 1.
Level of rnr message during cold shock and the stationary phase. Total RNA was extracted from CAN20-12E cells using the RNeasy extraction kit (Qiagen), and was quantitated by absorbance at 260 nm. The amount of rnr message was determined using dot blots (see Materials and Methods). (A) RNA from cells before and up to 60 min after cold shock at 10°C. The amount of rnr message prior to transfer to 10°C was set at 1. (B) Exponential phase cells (exp) were extracted when the culture reached an A550 of 0.3. Stationary phase cells (stat) were grown overnight. The data are the average of three experiments, which agreed almost exactly; the amount of rnr mRNA in the exponential phase has been set at 1.
FIGURE 2.
Stability of RNase R in the exponential phase. CAN20-12E cells were grown in YT medium, treated with chloramphenicol, and assayed for RNase R protein and activity (see Materials and Methods). (A) Representative blot of samples for RNase R and PNPase before and up to 60 min after addition of chloramphenicol. (B) Quantitation of four experiments, as shown in A, for RNase R and one experiment for PNPase. (Black bars) RNase R, (white bars) PNPase. (C) RNase R activity for a representative experiment up to 90 min after chloramphenicol addition. RNase R protein and activity at zero time of chloramphenicol addition were set at 100%.
FIGURE 3.
Stability of RNase R in the stationary phase. CAN20-12E cells were grown overnight in YT medium, treated with chloramphenicol, and assayed for RNase R protein and activity (see Materials and Methods). (A) Representative Western blot of samples before and up to 4 h after addition of chloramphenicol. (B) Quantitation of three experiments as shown in A. (C) RNase R activity for a representative experiment up to 270 min after chloramphenicol addition. RNase R protein and activity at zero time of chloramphenicol addition were set at 100%.
FIGURE 4.
Stability of RNase R during cold shock. CAN20-12E cells were grown to an A550 of ∼ 0.3 and transferred to 10°C. After 1 h at this temperature, one portion was withdrawn for analysis and a second portion was subjected to chloramphenicol treatment as in Figure 2. (A) Western blot of RNase R at 37°C, after 1 h at 10°C, and up to 20 h after chloramphenicol addition. (B) Quantitation of data in A. The amount of RNase R protein at 37°C was set to 100%.
References
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