Figure 1.
Downregulation of betT expression by RNase III.
(A) Effects of RNase III on steady-state levels of betT mRNA. Total RNA samples were prepared from the cultures of E. coli strains MG1655, MG1655rnc, and MG1655betT grown at 37°C in LB medium until an OD600 of 0.6 was reached. Steady-state levels of betT mRNA were determined by semi-quantitative RT-PCR analysis. (B) Effects of cellular RNase III concentration on betT mRNA decay demonstrated by northern blot analysis. E. coli strains MG1655 and MG1655rnc were grown as described above and total RNA samples were prepared from at 0 min, 0.5 min, 1 min, 2 min, and 4 min after the addition of rifampicin (1 mg ml−1). The relative amounts of betT mRNA transcripts were measured by setting the amount of betT mRNA in MG1655 cells (A) or in cultures 0 min after the addition of rifampicin (B) to 1, and plotted in the graphs. Amounts of full-length M1 RNA, which served to normalize the amount of betT mRNA in each RNA sample, were determined by semi-quantitative RT-PCR (A) or northern blot (B). The primers used for RT-PCR and PCR DNA for the production of random probe are shown with numbers indicating nucleotide positions in betT mRNA (the first nucleotide of the start codon as +1). The experiments were repeated three times and averaged. The error bars (standard errors of the mean) were used to indicate the range of assay results. The half-lives of betT mRNA were estimated by fitting and extrapolating the plots in the graphs.
Figure 2.
Identification of RNase III cleavage sites in betT mRNA in vitro and in vivo.
(A) In vitro cleavage of the full-length synthetic betT mRNA. The 5′-end-labeled betT transcript (4 pmol) was incubated with purified RNase III (1 pmol) in cleavage buffer with (+) or without (-) MgCl2 at 37°C. The size of the cleavage products was estimated using size markers generated by internally labeled transcripts. The major cleavage products are indicated with arrows. Other minor cleavage products are indicated with asterisks. (B) Primer extension analysis of betT mRNA. Total RNA was prepared from MG1655rnc harboring pBetRS1 and either pKAN6B or pRNC1, which exogenously overexpressed betT mRNA, hybridized with a 5′-32P-end-labeled primer (betT+120R), and extended using AMV reverse transcriptase. Sequencing ladders were produced using the same primer used in cDNA synthesis and PCR DNA, encompassing the betT gene as a template. (C) The predicted secondary structure of betT mRNA region encompassing RNase III cleavage sites. The secondary structure was determined using the M-fold program [33]. (D) In vitro cleavage of the model hairpin RNA of betT mRNA. 3′-end-labeled betT model hairpin (25 pmol) was incubated with purified RNase III (1 pmol) in a cleavage buffer with (+) or without (−) MgCl2, respectively. Cleavage products (I, II, III, and IV) were identified using size markers generated by alkaline hydrolysis and RNase T1 digestion. Relative abundance of each cleavage product was assessed by measuring the radioactivity of each band and plotted.
Figure 3.
Inhibition of RNase III cleavage of betT mRNA by introducing a mutation at the cleavage site.
(A) Secondary structures of the hairpin encompassing RNase III cleavage sites. Nucleotide substitutions (C33U and C39U) at the RNase III cleavage sites are indicated. (B) Effects of a nucleotide substitution at the cleavage sites on RNase III activity on betT mRNA. Primer extension experiments were performed as described in the legend to Figure 2B. (C) Effects of an RNase III cleavage site mutation on steady-state levels of betT mRNA determined by semi-quantitative RT-PCR analysis. Total RNA was prepared from strains MG1655rncbetT harboring pRNC1 and either pBetRS1 or its derivative (pBetRS1-C33U or pBetRS1-C39U), which were grown in LB at 37°C to an OD600 of 0.6.
Figure 4.
Osmoregulation of betT mRNA degradation by RNase III.
(A) Effects of betT deletion on E. coli growth upon osmotic stress. The cultures of wild-type and betT and/or proV-deleted MG1655 were grown in M63 supplemented with 22 mM glucose until an OD600 of 0.3 was reached, and either treated with 0.01 M (hypo-osmotic), 0.17 M (normal), or 0.50 M (hyper-osmotic) NaCl with 10 µM choline. Growth was measured by analyzing the cell density (OD600) of cultures grown for 18 h. (B) Effects of osmotic stress on the half-life of betT mRNA. Total RNA was isolated from the cultures of MG1655 and MG1655rnc grown under the same conditions as described above, except that they were grown until an OD600 of 0.6 was reached followed by the addition of rifampicin (1 mg ml−1). (C) Effects of the betT promoter on the half-life of betT mRNA. MG1655betT harboring pBetRS1 was grown as described above. Semi-quantitative RT-PCR analysis was performed to measure the relative amounts of betT mRNA. Experiments were performed in triplicate and repeated at least three times.