1566 Nucleic Acids Research, 2006, Vol. 34, No. 5 Figure 2. Suppression of the Pth(Ts) phenotype mediated by the over- production of tRNALys 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 (DvalU, DlysV; open symbols) or pVH119 (valU, lysV; closed sym- bols) 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 ¼ 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-tRNALys (closed circles), p-tRNAAsp1 (dark orange squares), p-tRNAAsn (blue triangles), p-tRNAArg4 (green diamonds), p-tRNAGly2 (pink pentagons) and p-tRNAVal1 (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-tRNAVal1 (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 tRNAVal1 (10). Therefore, the actual concen- tration of p-tRNAVal1 represented 30% of total tRNAVal1 at the non-permissive temperature. Concentration of Pth(Ts) protein Non-transformed and transformed strains of E.coli AA7852 were incubated at 32 C to an OD600 of 0.4 in liquid LB medium containing antibiotic as appropriate. Cultures were then incubated at 41 or 43 C and samples taken at the times indicated in Figure 1B. For each sample, cells were harvested by centrifugation, resuspended in lysis buffer [62.5 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol and 5% 2-mercaptoethanol] and boiled for 5 min. Total protein was resolved through 15% SDS–PAGE, and the gel electroblotted onto a nitrocellulose membrane. Pth(Ts) was detected by western blot using rabbit polyclonal anti-Pth serum (diluted 1:8000) (11), the signal being ampliﬁed by treatment with rabbit anti-IgA coupled to horseradish peroxidase (diluted 1:10,000) and revealed using Western blotting ﬂuorescent detection reagents (ECL, Amersham Pharmacia Biotech, Amersham, UK). Levels of aminoacyl- and peptidyl-tRNAs The levels of uncharged, aminoacylated and peptidylated tRNA were determined by northern blot assays as described previously (12). Liquid cultures (200 ml) of non-transformed and transformed strains of E.coli AA7852 were grown at 32 C to an OD600 of 0.2 in LB-Ap medium and then transferred to an incubator at 41 C. Samples (10 ml) were taken at the times indicated in Figures 2–4, the cells were pelleted at 4 C and total RNA isolated under acidic conditions (13). A portion of RNA was resuspended in 166 ml of diethyl pyrocarbonate- water and divided into two fractions, one of which, the control sample, was mixed with 6.7 ml of 3 M NaOAc (pH 5.0) and used to resolve the aminoacylated and peptidylated tRNA forms from the uncharged forms. The second sample was submitted to cupric ion-mediated hydrolysis in which aminoacylated tRNAs, but not the p-tRNA derivatives, were cleaved (14): this sample was mixed with 6.7 ml of 3 M NaOAc (pH 5.0) and 10 ml of 100 mM CuSO4. Each sample was incubated for 30 min at 37 C and adjusted to 5 mM with respect to Na2EDTA prior to precipitation with 2 vol of absolute ethanol at 20 C. Two additional control samples were prepared, in the presence or absence of 100 ng of a puriﬁed preparation of Pth(Ts) protein (Figure 3), each containing 3 mg of total tRNA (obtained 60 min after the temperature shift of the cells) dissolved in 25 ml of reaction buffer (Tris–HCl 100 mM and NH4Cl 100 mM) and 50 ml of a buffer containing 10 mM Tris–HCl (pH 7.6), 10 mM MgOAc, 6 mM b-mer- captoethanol and 20 mM NH4Cl. These samples were incubated for 20 min at 37 C and precipitated with 2 vol of absolute ethanol and 0.1 vol of 3 M NaOAc. The RNAs obtained from the untreated, the CuSO4 treated and the Pth treated samples were each dissolved in 5 ml of 10 mM NaOAc (pH 5.0) and 1 mM Na2EDTA, and an aliquot (1 ml) of each sample used to determine RNA concentration at 260 nm. Samples containing 4 mg of extracted RNA were resolved by overnight electrophoresis in a 6% polyacrylamide gel containing 8M urea in 0.07 M NaOAc buffer (pH 5.0) at 16 C. The RNA was electroblotted onto Hybond-N+ nylon membranes (Amersham Biosciences, Amersham, UK), in a BioRad (Hercules, CA) Trans-Blot Semi-Dry Electrophoretic Transfer Cell for 18 min at 380 mA using