An rne-1 pnp-7 double mutation suppresses the temperature-sensitive defect of lacZ gene expression in a divE mutant

Abstract

A divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, exhibits differential loss of the synthesis of certain proteins, such as beta-galactosidase and succinate dehydrogenase, at nonpermissive temperatures. In Escherichia coli, the UCA codon is recognized only by tRNA1Ser. Several genes containing UCA codons are normally expressed after a temperature shift to 42 degrees C in the divE mutant. Therefore, it is unlikely that the defect in protein synthesis at 42 degrees C is simply caused by a defect in the decoding function of the mutant tRNA1Ser. In this study, we sought to determine the cause of the defect in lacZ gene expression in the divE mutant. It has also been shown that the defect in lacZ gene expression is accompanied by a decrease in the amount of lacZ mRNA. To examine whether inactivation of mRNA degradation pathways restores the defect in lacZ gene expression, we constructed divE mutants containing rne-1, rnb-500, and pnp-7 mutations in various combinations. We found that the defect was almost completely restored by introducing an rne-1 pnp-7 double mutation into the divE mutant. Northern hybridization analysis showed that the rne-1 mutation stabilized lacZ mRNA, whereas the pnp-7 mutation stabilized mutant tRNA1Ser, at 44 degrees C. We present a mechanism that may explain these results.

FIG. 1

Induction of β-galactosidase in various divE mutants at 30 and 44°C. Cells were grown in M9 medium to mid-log phase at 30°C, and IPTG was then added to the culture at 2 mM. An aliquot of the culture was immediately shifted to 44°C, and another was grown at 30°C. One unit of enzyme activity was defined as that which hydrolyzed 1 μmol of ONPG per h. A, SKR207 (wild type); B, SKR319 (divE42); C, SKR101 (rne-1 pnp-7 rnb-500); D, SKR104 (rne-1 pnp-7 rnb-500 divE42). Symbols: ○, 30°C; •, 44°C. O.D._530nm; optical density at 530 nm.

FIG. 2

Northern hybridization analysis of lacZ mRNA. Cells were grown to mid-log phase at 30°C. IPTG was then added to the culture, and an aliquot was shifted to 44°C (lanes 2, 4, 6, and 8) while another aliquot was kept at 30°C (lanes 1, 3, 5, and 7). Samples were removed 30 min after the addition of IPTG. Total RNA (5 μg) was electrophoresed on a 1.5% formaldehyde-agarose gel and then blotted onto a nylon membrane. Hybridization was performed at 42°C by using the 300-bp fragment containing the lacZ gene as a probe. Lanes: 1 and 2, SKR207 (wild type); 3 and 4, SKR319 (divE42); 5 and 6, SKR101 (rne-1 pnp-7 rnb-500); 7 and 8, SKR104 (rne-1 pnp-7 rnb-500 divE42). Arrow, position of 3.1-kb lacZ mRNA. A 0.24- to 9.49-kb RNA ladder (GIBCO BRL) was used as a size marker. The values on the left are molecular sizes in kilobases.

FIG. 3

lacZ gene expression in the divE mutant containing an rne-1 mutation. Synthesis of β-galactosidase was measured as described in the legend to Fig. 1. A, SKR505 (rne-1); B, SKR615 (rne-1 divE42). Symbols: ○, 30°C; •, 44°C. C, Northern hybridization analysis of lacZ mRNA. For details, see the legend to Fig. 2. Lanes: 1, SKR505, 30°C; 2, SKR505, 44°C; 3, SKR615, 30°C; 4, SKR615, 44°C.

FIG. 4

Northern hybridization analysis of tRNA₁^Ser. Samples were removed as described for Fig. 2. Total RNA (2 μg) was electrophoresed on an 8% polyacrylamide gel containing 8 M urea and transferred onto a nylon membrane by electroblotting. A ³²P-labelled 359-bp fragment carrying the divE gene was used as a probe. Filled arrow, mature tRNA₁^Ser; open arrow, predicted nonprocessed tRNA₁^Ser. DNA fragments of pUC18 digested with HapII were used as molecular size standards. Lanes: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, 30°C; 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, 44°C. Strains used are shown at the top of the figure. The values on the right are molecular sizes in nucleotides.

FIG. 5

Degradation of ts-tRNA₁^Ser in divE mutants carrying various RNase mutations. Cells were grown to mid-log phase at 30°C. Rifampin was then added at 500 μg/ml, and an aliquot was shifted immediately to 44°C. Samples were removed at different time points from the culture at 30°C (○) or the culture at 44°C (•), and RNAs were extracted as described in Materials and Methods. Total RNA (2 μg) was electrophoresed on an 8% polyacrylamide–8 M urea gel and transferred onto a nylon membrane by electroblotting. A ³²P-labelled 359-bp fragment carrying the divE gene was used as a probe. The relative amount of mature tRNA₁^Ser was quantified with a BAS2000 Imaging Analyzer, expressed as a percentage of the value at the time of rifampin addition (time zero), and plotted as a function of time. A, SKR319; B, SKR615; C, SKR911.