1568 Nucleic Acids Research, 2006, Vol. 34, No. 5 Figure 4. The concentration of lysyl-tRNALys 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 (DvalU, DlysV) 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 tRNALys are shown. Experiments conducted with AA7852 cells transformed with pVH119 (valU, lysV) are indicated by closed symbols, whilst those that involved pVH124 (DvalU, DlysV) transformants are shown by open symbols: in each case p-tRNALys is represented by red squares, aminoacyl-tRNALys by blue circles, uncharged-tRNALys by green triangles, p-tRNAArg2 by black squares, aminoacyl-tRNAArg2 by cyan circles, and uncharged-tRNAArg2 by yellow tri- angles. The additional amount of tRNALys produced by the pVH119 transfor- mant was estimated with respect to the concentration of tRNAArg2, which was considered to be unaffected by the overproduction of tRNALys. Two successive hybridisations were conducted on the same membrane, first using a tRNALys probe and then with a tRNAArg2 probe, in order to normalize for the RNA load in different lanes. These data indicate that the total concentration of tRNALys in cells transformed with pVH119 was 2-fold higher than that in cells transformed with pVH124. The relative levels of the tRNALys forms in (B) have been multiplied by 2 to take this account. The data correspond to a representative experiment. from the restored capacity of the cells to grow at 41 C that the presence of Pth(Ts) protein at levels compatible with viability should continue indeﬁnitely. In AA7852 cells incubated at 32 C, neither the level (Figure 1B) nor the half- life of Pth(Ts) changed with tRNALys overproduction (data not shown). Thus, the overproduction of tRNALys may rise the rate of Pth(Ts) protein synthesis at a non-permissive temperature to levels that are just high enough to maintain the amount and activity of Pth(Ts) required for protein synthesis and cell viability. Overproduction of tRNALys slightly reduces the relative concentration of accumulated peptidyl-tRNAs in pth(Ts) cells at 41 C It was of interest to determine whether the steady-state level of Pth(Ts) mediated by the overproduction of tRNALys at a non-permissive temperature could relieve the accumulation of p-tRNA in the mutant cells. For this purpose the relative con- centrations of various p-tRNAs in pVH119-transformed AA7852 cells, incubated at 41 C, were compared with those found in AA7852 cells transformed with the pVH124 control construct. The results (Figure 2B and C) show that, after 60 min at 41 C, the relative concentrations of p-tRNAs of tRNALys, tRNAAsp1, tRNAAsn, tRNAVal1, tRNAArg4 and tRNAGly2 were consistently lower in pth(Ts) cells transformed with pVH119 compared with control transformants. In nearly all of the assays, the concentration of p-tRNA remained low for the ﬁrst few minutes after the cells had been transferred to 41 C, then increased for another 30 min, and began to level off after 60 min. This ﬁnding was conﬁrmed for all of the tRNAs analysed. It would thus appear that the limited levels of Pth(Ts) protein maintained during suppression of the effects of pth(Ts) mutation mediated by the overproduction of tRNALys can only partially handle the increased rate of accu- mulation of p-tRNA at 41 C. However, whilst the levels of p-tRNAs built up signiﬁcantly at 41 C, the cells were still able to grow although at a reduced rate in comparison with that observed at 32 C (Figure 2A). The complexity of the p-tRNALys increases with the time of incubation at 41 C The complexity of the p-tRNALys species that accumulated in the presence of limited levels of Pth(Ts) protein was evaluated by northern blots of samples extracted from pVH119- and pVH124-transformed AA7852 cells at different times after transference from 32 to 41 C. As shown in Figure 3A and B, during the ﬁrst 30 min at 41 C, the tRNALys species observed were mainly in the aminoacylated form. However, the level and the complexity of the accumulated p-tRNALys increased in a time-dependent manner as revealed by the increasing intensities and range of electrophoretic mobilities of the species above the free-tRNALys bands in the northern blots (Figure 3). This increase in accumulated p-tRNALys, irrespective of tRNALys overproduction, may result from an increased rate of translation at 41 C (3), given that the levels of Pth(Ts) protein in the overproducing cells were comparable at both 32 and 41 C (Figure 1B). Larger amounts of free-tRNA were generated by the cupric ion-mediated hydrolysis of RNA extracts obtained from pVH119-transformed pth(Ts) cells incubated at 41 C than from the pVH124-transformed strain