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Met for 10 min at 37°C, followed by addition of 150 pmol of 5′-Fe-BABE-ASL, then incubation for 20 min at 37°C and 20 min on ice. Hydroxyl radical strand scission was initiated as described (17). Extraction of rRNAs, primer extension, and gel electrophoresis were carried out as described (43). All of the observed Fe-BABE-dependent strand scissions were observed in at least three independent experiments and were quantified by Phosphor-Imager analysis (Molecular Dynamics).
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Phe. The predicted locations of the rRNA targets relative to the two tRNAs are independent of the arrangement of the tRNAs; the A-and P-site clouds are, in effect, attached to their respective tRNAs and will simply move along with them.
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Strong cleavages were defined as those that give rise to gel bands whose intensities are at least two-fold as strong as those of the nearby dideoxy sequencing bands; medium-strength cleavages, as those whose intensities are similar; and weak cleavages, as those whose intensities are half of or less than those of the sequencing bands. Target distances were calibrated in two independent ways. In one case, RNA-RNA duplexes were formed, in which the 5′ end of one strand was derivatized with the Fe(II) probe, and cleavage of the opposite strand was monitored by primer extension. The other approach is an internal calibration inherent in the ASL cleavage data themselves; a lower limit of the probing range for a strong hit at a given target site is half the distance between the two most widely separated ASL tethering sites from which strong cleavage is observed. For example, the maximum separation observed between probe positions for two strong cleavages at the same target is position 1924 by ASL6 and ASL10 (Fig. 3G), corresponding to a distance of about 20 Å between the corresponding ASL phosphate positions. The distance to the target can be estimated as half this distance (10 Å) plus or minus the length of the tether arm (12 Å). This gives a distance range of 0 to 22 Å between the tethering position and target position for strong cleavages. By similar reasoning, we estimate medium-strength cleavages to indicate a range of 12 to 36 Å, and weak cleavages, a range of 20 to 44 Å. The data obtained by means of the duplex method fall into these overlapping ranges, providing independent validation of the values derived from internal calibration. These values are also in good agreement with those reported in previous studies (27).
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We thank C. R. Woese for suggesting the internal calibration strategy; D. P. Greiner, J. Moran, and C. F. Meares for helping with synthesis of BABE and for providing samples of BABE used in early experiments; members of the Puglisi lab for providing T7 RNA polymerase; M.I. Recht for help with EXCEL; K. Lieberman and R. Green for discussions; and J. Cate, G. Culver, L. Newcomb, R. Green, K. Wilson, K. Lieberman, and L. Holmberg for review of the manuscript. Supported by grant GM-17129 from the NIH and from a grant to the Center for Molecular Biology of RNA from the Lucille P. Markey Charitable Trust.
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