(Figure 3A and B, compare the pairs of 60 min). RNA samples, obtained 60 min after the temperature shift of the pth(Ts) transformed cells, were treated with a puriﬁed preparation of Pth protein (Figure 3, right panels). It was observed that the tRNALys forms that had been concentrated in the smear localized above the aminoacylated form disappeared, whilst the levels of free-tRNALys increased proportionally (Figure 3A and B, right panels) indicating the p-tRNA nature of the material in the smear. The faint band of aminoacylated- tRNALys (Figure 3A, right panel, +Pth lane) did not corre- spond with the relatively intensity material produced by cupric ion-mediated hydrolysis of the 60 min sample (Figure 3A, left panel), although both bands should represent the same proportion of aminoacylated-tRNALys. Since this was not the case, it is likely that part of the aminoacylated-tRNA was hydrolysed during the preparation of samples for treatment with Pth. Overproduction of tRNALys increases the levels of aminoacyl-tRNALys Since the levels of aminoacylation determine the availability of precursors for protein synthesis in the cell, it was important to assess the extent by which transformation of pth(Ts) cells with pVH119 changed the concentrations of the tRNALys species present. For this purpose, the levels and relative proportions of tRNALys found as uncharged-, aminoacyl- and p-tRNA were determined in pth(Ts) cells harbouring pVH119 and pVH124 constructs (Figure 4). Whilst the level of total tRNALys detected in pVH119-transformed cells was 2-fold greater than that found in the control trans- formants at 32 C (data not shown) (7), the relative proportions of the different tRNALys species and of the control tRNAArg2 were comparable in both cell lines (Figure 4A). Following the temperature rise to 41 C, the levels of aminoacylated tRNALys and tRNAArg2 in both pVH119- and pVH124-transformed cells decreased with time, whilst the amounts of the respective p-tRNAs increased proportionally (Figure 4B and C). Taking into consideration that the levels of both lysyl-tRNALys and p-tRNALys increased 2-fold in the pVH119-transformed cells, the suppression of the temperature-sensitivity of the pth(Ts) strain may be a consequence of an increase in the absolute concentration of lysyl-tRNALys rather than of a change in the concentration of an inhibitory p-tRNA (5). DISCUSSION In this work we showed that the overproduction of tRNALys was able to suppress the thermosensitivity of E.coli strain AA7852, the original pth(Ts) isolate (1), at 41 C but not at 43 C, a result which differs from that obtained previously for C600 pth(Ts) in which suppression was observed even at 43 C (7). Suppression of the effects of pth(Ts) mutation was accom- panied by a rise in the rate of Pth(Ts) protein synthesis that was clearly just sufﬁcient to maintain cell viability at 41 C. Fur- thermore, the levels of Pth(Ts) protein maintained by tRNALys overproduction permitted linear growth of the mutant cells at 41 C (Figure 2A), but not exponential growth as would be expected if the mutation were fully suppressed. The levels of Pth(Ts) protein at 41 C were comparable with those present in pth(Ts) cells at 32 C (compare t ¼ 0 and 120 min lanes in Nucleic Acids Research, 2006, Vol. 34, No. 5 1569 Figure 1B, e) namely, some 5-fold lower than the wild-type concentration (3). The maintenance of the concentration of Pth(Ts) protein in the presence of tRNALys overproduction was not due to an extended stability of the protein (data not shown), although an effect on the levels of pth(Ts) mRNA cannot be ruled out owing to the difﬁculty in assaying the low concentrations of mRNA in untransformed cells. It is argued that the higher availability of charged-tRNALys, rather than the reduced level of p-tRNALys, provokes a general enhancement in protein synthesis that maintains low but effec- tive concentrations of Pth(Ts) protein at 41 C. The results showed that the restoration of growth of the pth(Ts) mutant at a restrictive temperature mediated by the overproduction of tRNALys was correlated with signiﬁcant increases in the levels of both charged-tRNALys and p-tRNALys (Figure 4B). If p-tRNALys were poisoning translation one would not expect a higher level to be correlated with the observed restoration of growth. The concentration of lysyl-tRNALys in pVH119-transformed cells decreased with the time of incuba- tion at 41 C (Figure 4B); however, the proportion of this species with respect to the total tRNALys always remained higher than that present in the pVH124-transformants. The fractions of the aminoacylated-tRNAArg2 species decreased in both pVH119- and pVH124-transformants after transfer of the cells to 41 C (Figure 4C). After 60 min incubation, however, the relative concentration of aminoacylated- tRNAArg2 in pVH119-transformed cells was somewhat higher than in the pVH124-transformed counterparts, apparently at the expense of the p-tRNAArg2 fraction. Also, the pth(Ts) transformant that overproduced tRNALys contained 20–50% less p-tRNA of other species than the non-overproducing con- trol cells after 2 h incubation at 41 C (Figure 2B and C). This behaviour may be explained by assuming that the overproduc- tion of tRNALys maintains the levels of Pth(Ts) protein by enhancing its synthesis. As a result Pth(Ts) would hydrolyse the p-tRNAs thus raising the amounts of other aminoacylable tRNAs. The limited increase in the relative concentrations of the charged tRNAs may help in maintaining the cell viability indeﬁnitely. The proportion of charged-tRNALys to free-forms remained unchanged following transformation of pth(Ts) cells with pVH119 and further growth at 32 C, in spite of a 2-fold increase in the levels of tRNALys (see legend to Figure 4) (7). However, upon incubation at 41 C, the concentration of charged-tRNALys fell gradually over 60 min to levels com- parable with those observed in the control cells incubated at 32 C, (Figure 4B, compare overproduced tRNALys at 60 min with control at time 0 min). Apparently, once this critical phase of 60 min is overcome, the high levels of Lys-tRNALys mediated by tRNALys overproduction are not essential for cell viability. This result suggests that the surplus charged-tRNALys overcomes the transient effect of the heat shock response on protein synthesis that is occasioned by a temperature shift to 41 C. The increasing intensity and extent of the smear located above the free-tRNA bands in the northern blots (Figure 3) suggested that the concentration of the accumulated material and the apparent complexity of the p-tRNALys species increased after the ﬁrst 30 min following transfer of the cells to 41 C. Analogous patterns of change with respect to time were observed for the p-tRNA derivatives of tRNAArg2,