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. 2003 Jan 15;31(2):596–601. doi: 10.1093/nar/gkg131

Figure 2.

Figure 2

(A) PAGE autoradiogram for DEPC chemical probing experiments with WT, U3271C and A3261G/U3271C hs mt tRNALeu(UUR). Lanes 1–3 (labeled WT, U3271C and A3261G/U3271C) are control lanes for the 5′-radiolabeled hs mt tRNALeu(UUR) samples. Lane G is a T1 digest of denatured WT hs mt tRNALeu(UUR). Lanes 5–7 are chemical probing reactions performed under native conditions. Samples were analyzed by 15% PAGE. The numbering of the bands corresponds to the positions of adenosine within the hs mt tRNALeu(UUR) sequence. Increased nuclease susceptibility was observed reproducibly for the U3271C mutant, as evidenced by the larger intensities of the bands in the control lane for this sample. (B) Analysis of chemical probing. The relative percent cleavage at the various adenine positions was quantitated (Image Quant) by first normalizing the values against the uncleaved tRNA within each lane to adjust the values for loading variations. The cleavage intensities at each adenine position for the mutant tRNAs were compared with those of the WT sample. Position 30 within the A3261G/U3271C mutant sequence is not an adenine. Data were obtained from greater than three independent trials; protection values from different trials varied ±0.02.