Nucleic Acids Research, 2006, Vol. 34, No. 5 1567 Figure 3. Suppression of the Pth(Ts) phenotype mediated by the overproduction of tRNALys allows the accumulation of heterogeneous p-tRNALys at 41 C. Changes in the concentrations of the tRNALys 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 (DvalU, DlysV)] of excess tRNALys are presented. Transformed cells were grown at 32 C to an OD600 of 0.2 prior to transfer at time ¼ 0 min to 41 C, and samples were taken at the times indicated. p-tRNALys 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 tRNALys derivatives are arrowed, whilst each brace indicates the extent of the smear corresponding to heterogeneous p-tRNALys. 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). 1· TAE as transfer buffer. tRNA-speciﬁc oligodeoxyribonu- cleotide probes were synthesized according to a published method (15). The blotted membranes were pre-hybridized with denatured salmon sperm DNA for 3 h at 42 C, and hybridized to 50-32P end-labelled oligonucleotides for 16 h at 42 C. Finally, the membranes were washed twice for 1 h at room temperature (13) and the radioactive signals quantiﬁed using a Typhoon Scan (Amersham Biosciences). The mem- branes were routinely probed at least twice with other oligonu- cleotides. The respective percentages of uncharged, aminoacylated or peptidylated tRNAs were estimated relative to the total tRNA for each electrophoretic lane using the following formula: % tRNA species ðuncharged-‚ aminoacyl -or peptidyl-Þ tRNA species · 100 ¼ ðuncharged tRNA þ aminoacylated tRNA þ peptidylated tRNAÞ : RESULTS Overproduction of tRNALys suppresses the effects of pth(Ts) mutation at 41 C but not at 43 C E.coli strain AA7852 was transformed with the construct pVH119, harbouring the gene lysV encoding tRNALys, and control constructs pVH124 and pVH125 in which lysV was either partly or fully deleted (7). Growth assays, performed by streaking independent colonies on LB-Ap medium (Figure 1A), demonstrated that cells containing pVH119 exhibited signiﬁcant suppression of the Pth(Ts) phenotype at 41 C, but much lower growth at 43 C. In contrast, there was hardly any detectable growth of cells containing the control constructs at either of these temperatures. Overproduction of tRNALys maintains the levels of Pth(Ts) protein at 41 C E.coli strain AA7852 was transformed with constructs harbouring different tRNA-encoding genes and incubated at 41 or 43 C: the concentrations of Pth(Ts) protein were deter- mined in extracts of these cells by western blot analysis (Figure 1B). The control construct pVH124, and those bearing the tRNA-encoding genes valU, argX, hisR, leuT, proM and thrW, were ineffective in suppressing the thermosensitivity of the pth(Ts) strain at 41 C (data not shown) as has been demonstrated previously (7). Accordingly, these constructs were unable to maintain detectable levels of Pth(Ts) antigen at 41 C (Figure 1B, a–d). However, overproduction of tRNALys gave rise to levels of Pth(Ts) antigen that were clearly detectable for at least 2 h following transfer of the cells to 41 C, but only for the ﬁrst 30 min or so after cells were transferred to 43 C (Figure 1B, e). It may be inferred