Excess of charged tRNALys maintains low levels of peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature

Abstract

Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNA(Lys), of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41 degrees C whereas plasmids bearing other tRNA genes were ineffective. At 32 degrees C the excess of tRNA(Lys) did not alter the percentages of the free-, charged- or peptidyl-tRNA(Lys) species compared with those found in strains that did not overproduce tRNA(Lys). At 41 degrees C, however, despite increases in the level of peptidyl-tRNA(Lys), the excess tRNA(Lys) helped to maintain the concentration of charged-tRNA(Lys) at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNA(Lys) at 41 degrees C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNA(Lys) and the causes of tRNA(Lys) starvation in pth(Ts) strains grown at non-permissive temperatures are considered.

Figure 1

Suppression of the Pth(Ts) phenotype mediated by the overproduction of tRNA^Lys maintains moderate levels of the Pth(Ts) protein. (A) Depicts the cellular growth of the pth(Ts) mutant strain AA7852 separately transformed with pVH124 (ΔvalU, ΔlysV), pVH125 (valU, ΔlysV) or pVH119 (valU, lysV) incubated at different temperatures. Isolated colonies of the independent transformants were streaked onto LB-Ap plates and incubated overnight at the indicated temperatures. (B) Presents the immunodetection of Pth(Ts) in the pth(Ts) mutant strain AA7852 separately transformed with pVH124, pVH125, pVH119, ptRNA^HisCCA (argX, hisR, leuT, proM) or pTH2 (thrW) and grown at 32°C prior to transfer at time = 0 min at 41 or 43°C. The concentration of Pth(Ts) protein was estimated by immunoblot analysis. The left lane shows purified wild-type Pth protein, which migrates slightly faster in SDS–PAGE than the Pth(Ts) variant (arrowed) (3).

Figure 2

Suppression of the Pth(Ts) phenotype mediated by the overproduction of tRNA^Lys promotes growth and reduces only moderately the concentration of p-tRNAs at 41°C. (A) Presents the growth in liquid medium of the pth(Ts) mutant strain AA7852 separately transformed with pVH124 (ΔvalU, ΔlysV; open symbols) or pVH119 (valU, lysV; closed symbols) at 32 and 41°C. Cells were grown at 32°C to an OD₆₀₀ of 0.2 and further incubated at 32°C (grey symbols) or transferred to 41°C at time = 0 min (orange symbols). (B and C) show the percentages of p-tRNAs accumulated during growth at 41°C as described in (A). The relative fractions of the various p-tRNAs [p-tRNA^Lys (closed circles), p-tRNA^Asp1 (dark orange squares), p-tRNA^Asn (blue triangles), p-tRNA^Arg4 (green diamonds), p-tRNA^Gly2 (pink pentagons) and p-tRNA^Val1 (yellow hexagons)] were quantified by northern blotting of acid urea gels. The curves show representative experiments. Note: half of the material extracted from the cells that banded as aminoacyl- or p-tRNA^Val1 (yellow hexagons) in the acid urea gels was refractory to treatments with purified Pth protein or with CuSO₄ (data not shown) and may correspond to a partially modified variant of tRNA^Val1 (10). Therefore, the actual concentration of p-tRNA^Val1 represented 30% of total tRNA^Val1 at the non-permissive temperature.

Figure 3

Suppression of the Pth(Ts) phenotype mediated by the overproduction of tRNA^Lys allows the accumulation of heterogeneous p-tRNA^Lys at 41°C. Changes in the concentrations of the tRNA^Lys forms with time in the presence (A) [showing strain AA7852 transformed with pVH119 (valU, lysV)] or in the absence (B) [showing strain AA7852 transformed with pVH124 (ΔvalU, ΔlysV)] of excess tRNA^Lys are presented. Transformed cells were grown at 32°C to an OD₆₀₀ of 0.2 prior to transfer at time = 0 min to 41°C, and samples were taken at the times indicated. p-tRNA^Lys was revealed by northern blotting using a specific ³²P-labelled oligo-probe in samples of total RNA. The sample in the right lane was treated with a preparation of Pth protein prior to electrophoresis. The relative locations of the various tRNA^Lys derivatives are arrowed, whilst each brace indicates the extent of the smear corresponding to heterogeneous p-tRNA^Lys. The absolute amount of material applied to each lane varied according to the efficiency of recovery of the tRNA in each manipulation. Band intensities of the different tRNA forms should, strictly, only be compared within lanes, thus only approximate indications of amounts of tRNA forms can be derived between independent lanes (compare 30 min lanes treated and untreated with CuSO₄).

Figure 4

The concentration of lysyl-tRNA^Lys in transformed pth(Ts) cells decreases during incubation at 41°C. (A) Presents the relative amounts of aminoacyl-, peptidyl- and uncharged-tRNA in strain AA7852 transformed with pVH124 (ΔvalU, ΔlysV) or pVH119 (valU, lysV) and incubated at 32°C. The percentage of each of the tRNA forms relative to total tRNA (uncharged plus aminocylated plus peptidylated tRNA) was estimated by comparing the signal intensities before and after treatment with CuSO₄ (Figure 3). In (B and C), changes in the relative fractions of the Lys and Arg2 tRNA forms, respectively, during suppression of the Pth(Ts) phenotype mediated by the overproduction of tRNA^Lys are shown. Experiments conducted with AA7852 cells transformed with pVH119 (valU, lysV) are indicated by closed symbols, whilst those that involved pVH124 (ΔvalU, ΔlysV) transformants are shown by open symbols: in each case p-tRNA^Lys is represented by red squares, aminoacyl-tRNA^Lys by blue circles, uncharged-tRNA^Lys by green triangles, p-tRNA^Arg2 by black squares, aminoacyl-tRNA^Arg2 by cyan circles, and uncharged-tRNA^Arg2 by yellow triangles. The additional amount of tRNA^Lys produced by the pVH119 transformant was estimated with respect to the concentration of tRNA^Arg2, which was considered to be unaffected by the overproduction of tRNA^Lys. Two successive hybridisations were conducted on the same membrane, first using a tRNA^Lys probe and then with a tRNA^Arg2 probe, in order to normalize for the RNA load in different lanes. These data indicate that the total concentration of tRNA^Lys in cells transformed with pVH119 was 2-fold higher than that in cells transformed with pVH124. The relative levels of the tRNA^Lys forms in (B) have been multiplied by 2 to take this account. The data correspond to a representative experiment.