260909.fig.003a
(a)
260909.fig.003b
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260909.fig.003c
(c)
Figure 3: Val-tRF associates with ribosomes in vitro and in vivo. (a) A representative polysome gradient of H. volcanii. Fractions containing polysomes, 50S, or 30S subunits were collected and used for northern blot analyses. The identity of the individual fractions was confirmed by agarose gel electrophoresis. (b) The presence of the Val-tRF in the different gradient fractions was investigated by northern blot analysis using RNA from unstressed cells or from cultures incubated at high pH (pH 8.5). Arrows indicate the full-length Val-tRNA and the 26 nt long fragment detected in the cDNA library. (c) In vitro filter binding studies of radiolabelled synthetic Val-tRF on ribosomal particles (70S, 50S, 30S) from H. volcanii (left panel). As a positive control, binding of to E. coli 70S was monitored. To confirm specific binding of Val-tRF an equally long fragment of isoleucine tRNA (Ile-tRF), an RNA sequence not found in our cDNA screen, served as negative control. (Right panel) Quantification of relative binding whereas association of Val-tRF to 70S was normalized to 100%. Signals measured in the absence of any ribosomal particles (-) were subtracted from all experimental points. Error bars show the mean and standard deviation of at least four independent experiments.