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. 2014 Jun 26;42(13):8621–8634. doi: 10.1093/nar/gku539

Figure 4.

Figure 4.

Protections against chemical probes in nucleotides of the central loop of domain V of 23S rRNA, caused by binding of PA–CAM conjugates (compounds 4 and 5) to Escherichia coli ribosomes. Ribosomes were incubated in the presence or absence of each PA–CAM conjugate at 25°C for 2 s or 3 min. The resulting complexes were then probed with DMS (A), CMCT (B) or Kethoxal (C). U, A, G and C, dideoxy sequencing lanes; lanes 1 and 5, unmodified ribosomes; lanes 2 and 6, ribosomes probed in the absence of PA–CAM conjugates; lanes 3 and 7, ribosomes pre-incubated with each PA–CAM conjugate for 2 s and then probed; lanes 4 and 8, ribosomes pre-incubated with each PA–CAM conjugate for 3 min and then probed. Results obtained with CAM, although published previously (4), were reproduced, and are presented in lanes 9–11 for the sake of comparison. Numbering of nucleosides for the sequencing lanes is indicated at the left. Nucleosides with accessibility affected by bound PA–CAM conjugate are shown by arrows at the right, while reference bands whose intensity is not affected by PA–CAM conjugates or CAM binding are indicated by an asterisk. The relative intensity of each reference band between lanes was used to correct the variability between lanes (horizontal normalization).