The SgrS-mediated repression of IICB^Glc occurs without recruitment of RNase E to the ptsG mRNA. (A) Effect of short-term phosphosugar stress on ptsG mRNA. TM338 (rne-FLAG), TM339 (Δpgi rne-FLAG), TM528 (rne701-FLAG), and TM525 (Δpgi rne701-FLAG) were grown in LB medium to A₆₀₀ = 0.5. To each culture, 0.4% glucose was added, and incubation was continued for 20 min. Total RNAs were prepared, and each RNA sample (10 or 5 μg) was subjected to Northern blotting with ptsG or sgrS probes. (B) Effect of long-term phosphosugar stress on ptsG mRNA. Cells were grown in LB medium to A₆₀₀ = 0.1. To each culture, 0.4% glucose was added, and incubation was continued for 2 h. Total RNAs were prepared, and each RNA sample (10 μg) was subjected to Northern blotting with ptsG probe. (C) Effect of long-term phosphosugar stress on IICB^Glc expression. Cells were grown in LB medium to A₆₀₀ = 0.1. To each culture, 0.4% glucose was added, and incubation was continued for 2 h. Crude extracts were prepared, and each protein sample corresponding to 0.04 A₆₀₀ was loaded on the gel and subjected to Western blotting with anti-IIB^Glc antibody.

The TM151 (ams1) cells harboring pPtsG-FLAG were grown to A₆₀₀ = 0.4 at 30°C in M9–0.4% glycerol medium without methionine. To each culture, 0.01% glucose or αMG was added, and incubation was continued for 15 min at 42°C. Each culture was exposed to [³⁵S]methionine for 1 min and subjected to an IP experiment as described in Materials and Methods. (A) The crude extract (Total; lanes 1 and 2) and bound fraction (IP; lanes 3 and 4) corresponding to 0.04 or 0.12 A₆₀₀ unit, respectively, were analyzed by 12% SDS/PAGE followed by autoradiography. (B) Total RNAs were prepared before the treatment with [³⁵S]methionine. Each RNA sample (0.5 or 5 μg) was subjected to Northern blotting with ptsG or sgrS probe.

Effect of C-terminal truncation of RNase E and the sgrS deletion on the cell growth. Overnight cultures of TM339 (Δpgi rne-FLAG), TM525 (Δpgi rne701-FLAG), and TM671 (Δpgi rne-FLAG ΔsgrS) were 100-folded diluted and grown in LB medium containing 0.4% glucose. Cell growth was monitored by measuring OD at 600 nm.

Model for gene silencing by sRNP. (Left) Ribonucleoprotein complex consisting of Hfq, RNase E, and SgrS or RyhB (sRNP) acts on the target mRNA through base-pairing, resulting in translational repression and rapid mRNA degradation that requires RNase E within sRNP. (Right) Ribonucleoprotein complex lacking RNase E could act on the target mRNA to cause translational inhibition without the rapid degradation of mRNA. The translational inhibition may lead to the gradual destabilization of target mRNAs that does not depend on RNase E within sRNP.

SgrS inhibits the translation of IICB^Glc in the absence of the RNase E-dependent rapid degradation of ptsG mRNA. TM641 (nre-HA), TM642 (rne701-HA), and TM666 (rne-HA ΔsgrS) harboring pPtsG-FLAG were grown until A₆₀₀ = 0.4 in M9–0.4% glycerol medium without methionine. To each culture, 0.01% glucose or αMG was added, and incubation was continued for 10 min. (A) Each culture was exposed to [³⁵S]methionine for 1 min and subjected to IP experiments as described in Materials and Methods. The crude extract (Total; lanes 1–6) and bound fraction (IP; lanes 7–12) corresponding to 0.04 or 0.12 A₆₀₀ unit, respectively, were analyzed by 12% SDS/PAGE followed by autoradiography. (B) Total RNAs were prepared before the treatment with [³⁵S]methionine. Each RNA sample (0.5 or 5 μg) was subjected to Northern blotting with ptsG or sgrS probe.

Effect of kasugamycin on the rapid degradation of ptsG mRNA. Wild-type IT1568 cells were grown in LB medium to OD₆₀₀ = 0.3. Cells were exposed to kasugamycin (300 μg/ml) for 30 min, and then to either 0.4% glucose or αMG for 15 min. Total cellular RNAs were isolated and subjected to Northern blot analysis by using the ptsG or sgrS probe (lanes 3 and 4). Total cellular RNAs were also isolated from cells exposed to glucose or αMG without kasugamycin treatment and subjected to Northern blot analysis (lanes 1 and 2). Each RNA sample (10 or 5 μg) was used to detect ptsG or sgrS mRNA.

RyhB inhibits the translation of SodB in the absence of the RNase E-dependent rapid degradation of sodB mRNA. TM641 (nre-HA), TM642 (rne701-HA), and TM667 (rne-HA ΔryhB) harboring pTM31 (pSodB-Met₂-FLAG) were grown until A₆₀₀ = 0.4 in M9–0.4% glycerol medium containing 10 μM FeSO₄ without methionine. Then, 250 μM 2,2′-dipyridyl was added to each culture, and incubation was continued for 10 min. (A) The cellular RNAs were prepared, and each RNA sample (0.5 or 5 μg) was subjected to Northern blotting with sodB or ryhB probe. (B) Each culture was treated with [³⁵S]methionine for 1 min and subjected to IP experiments as described in Materials and Methods. The crude extract (Total; lanes 1, 2) and bound fraction (IP; lanes 3, 4) corresponding to 0.02 and 0.04 A₆₀₀ unit, respectively, were analyzed by 15% SDS/PAGE followed by autoradiography.