Enhancement of the synthesis of RpoE and StpA by polyamines at the level of translation in escherichia coli under heat shock conditions

Abstract

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified with a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate at 42 degrees C. Polyamines had a greater effect on cell growth at 42 degrees C than at 37 degrees C. At 42 degrees C, the synthesis of RpoE (sigma(24)) and StpA, which are involved in the transcription of a number of heat shock response genes, was stimulated by polyamines at the level of translation. In the rpoE and stpA mRNAs, a Shine-Dalgarno (SD) sequence is located at 13 and 12 nucleotides, respectively, upstream of the initiation codon AUG. When the SD sequences were moved to the more common position 7 nucleotides upstream of the initiation codon AUG, the degree of polyamine stimulation was reduced, although the level of RpoE and StpA synthesis was markedly increased. The mechanism underlying polyamine stimulation of RpoE synthesis was then studied. Polyamine stimulation of RpoE synthesis was reduced by changing the bulged-out structure in the initiation site of rpoE mRNA, although the level of RpoE synthesis increased. A selective structural change of this bulged-out region induced by spermidine at 42 degrees C was observed by circular dichroism. Polyamine stimulation of fMet-tRNA binding to ribosomes at 42 degrees C also disappeared by changing the bulged-out structure in the initiation site of rpoE mRNA. The results suggest that polyamines enhance the synthesis of RpoE by changing the bulged-out structure in the initiation site of rpoE mRNA.

FIG. 1.

Growth and polyamine content of E. coli MA261 at 37°C and 42°C. (A) Culture of E. coli MA261 cells with or without 100 μg/ml putrescine. (B) Polyamine content in cells harvested at an A₅₄₀ of 0.15 was measured as described in Materials and Methods. Data are shown as means ± standard errors. PUT, putrescine; SPD, spermidine.

FIG. 2.

Effect of polyamines on synthesis of RpoE in E. coli MA261 cells at 42°C. (A) Cells were harvested at an A₅₄₀ of 0.15. Western blotting of RpoE was performed using 20 μg of protein of cell lysate. (B) Dot blot and Northern blot analyses of rpoE mRNA were performed as described in Materials and Methods. (C) Schematic of the rpoE-lacZ fusion genes. The rpoE gene containing a 203-nucleotide 5′-upstream region with an unmodified or modified SD sequence and a 184-nucleotide open reading frame was fused to the lacZ gene. (D) Western blotting of RpoE-β-Gal fusion protein was performed using 10 μg of protein of cell lysate. (E) Immunoprecipitation of [³⁵S]methionine-labeled protein by antibody against β-Gal was performed as described in Materials and Methods. Values are means ± standard errors of triplicate determinations. Student's t test was performed for the value obtained in the presence of putrescine versus that in the absence of putrescine. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01. PUT, putrescine.

FIG. 3.

Levels of seven sigma factors and proteins encoded by polyamine modulons in E. coli MA261 cells at 42°C. Cells were harvested at an A₅₄₀ of 0.15. (A) Western blotting was performed using 10 μg protein of cell lysate for RpoD; 20 μg protein of cell lysate for RpoN, RpoS, RpoH, RpoF, and RpoE; and 50 μg protein of cell lysate for FecI. (B) Western blotting was performed using 1 μg of protein of cell lysate for OppA and 20 μg of protein of cell lysate for Cra, RF2, Fis, H-NS, and Cya. (C) Schematic of control of transcription of rpoH by RpoD and RpoE. (D) Dot blot and Northern blot analyses of rpoH mRNA were performed as described in Materials and Methods. Values are means ± standard errors of triplicate determinations. ns, P > 0.05; **, P ≤ 0.01. PUT, putrescine.

FIG. 4.

Effect of polyamines on synthesis of StpA in E. coli MA261 cells at 42°C. (A) Cells were harvested at an A₅₄₀ of 0.15. Western blotting of StpA was performed using 20 μg of protein of cell lysate. (B) Dot blot and Northern blot analyses of stpA mRNA were performed as described in Materials and Methods. (C) Schematic of the stpA-lacZ fusion genes. The stpA gene containing a 220-nucleotide 5′-upstream region with an unmodified or modified SD sequence and a 106-nucleotide open reading frame was fused to the lacZ gene. (D) Western blotting of StpA-β-Gal fusion protein was performed using 10 μg of protein of cell lysate. (E) Immunoprecipitation of [³⁵S]methionine-labeled protein by antibody against β-Gal was performed as described in Materials and Methods. Values are means ± standard errors of triplicate determinations. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01. PUT, putrescine.

FIG. 5.

Effect of polyamines on the synthesis of RpoE-β-Gal fusion protein derived from mutated rpoE-lacZ mRNA in the 5′ untranslated region. (A) Possible secondary structure of the initiation region of rpoE mRNA was constructed by the method of Zuker (49). The positions in which the sequence was mutated are shown. (B) Effect of polyamines on RpoE-β-Gal fusion protein synthesis was tested by Western blot analysis using 10 μg of protein of cell lysate. The levels of rpoE-lacZ mRNA in cells cultured with or without 100 μg/ml putrescine were nearly equal, judging from dot blot analysis. (C) Immunoprecipitation of [³⁵S]methionine-labeled protein by antibody against β-Gal was performed as described in Materials and Methods. Values are means ± standard errors of triplicate determinations. Δ, removal of the nucleotide; +, addition of the nucleotide; →, the replacement of the nucleotide; ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; PUT, putrescine.

FIG. 6.

CD spectra of RpoE WT RNA and RpoE −20(ΔU) RNA. (A and E) Structure of RpoE WT RNA and RpoE −20(ΔU) RNA. (B and F) CD spectra were recorded as described in Materials and Methods. Green line, no addition; blue line, 1.6 mM Mg²⁺; red line, 1.6 mM spermidine; black line, 1.6 mM Mg²⁺ and 1.6 mM spermidine. (C and G) Concentration-dependent shifts induced by Mg²⁺ at 37°C, Mg²⁺ at 42°C, spermidine at 37°C, or spermidine at 42°C in magnitude at 208 nm are shown. Values are means ± standard deviations for three determinations. (D and H) The K_dapp values of spermidine for RpoE WT RNA and RpoE −20(ΔU) RNA at 37°C and 42°C were determined according to the double-reciprocal equation plot. SPD, spermidine.

FIG. 7.

Effect of spermidine on RpoE WT (166-mer) and RpoE −20(ΔU) (165-mer) RNA-dependent [³H]fMet-tRNA binding to ribosomes. The binding of [³H]fMet-tRNA to ribosomes in the presence of RpoE WT RNA (A) or RpoE −20(ΔU) RNA (B) was measured in the presence of various concentrations of Mg²⁺ with (•) or without (○) 1 mM spermidine at 37°C or 42°C. (C) The binding of [³H]fMet-tRNA to ribosomes with RpoE WT RNA (•) or RpoE −20(ΔU) RNA (□) was measured in the presence of various concentrations of spermidine and 3 mM Mg²⁺ at 42°C. Values are means ± standard errors of triplicate determinations. SPD, spermidine.