Hydroxyl footprinting

Hampel and Burke observed that protection of hammerhead backbone sites in Mg + solutions required assembly of the full ribozyme-substrate complex. In other words, testing of ribozyme or substrate separately in the hydroxyl footprinting assay showed essentially complete hydrolysis of all nucleotides (Figure 2B of reference 56). In contrast, the fully assembled ribozyme-substrate complex showed protection of nucleotides structurally near the densely packed three-helix junction of hammerhead constructs HH16, HHal, and RNA 6. Two of the ribozyme group of protected nucleotides (Gs, Ae) are part of the conserved uridine U-turn seen in all known hammerhead constructs. (See Figures 6.10,6.11, and 6.12.) The footprinting results are collected in Table 6.5. 

TABLE 6.5 Protected Nucleotides in Hydroxyl Footprinting Assay as Adapted from Reference 57... 

Rothmel, RK, DL Shinbarger, MR Parsek, TL Aldrich, AM Chakrabarty (1991) Functional analysis of the Pseudomonas putida regulatory protein CatR transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting. J Bacterial 173 4717-4724. 

Tullius, T.D., and Dombroski, B.A. (1986) Hydroxyl radical footprinting high-resolution information about DNA-protein contacts and application to repressor and Cro protein. PNAS 83, 5469-5473. 

In one of the earlier reports hydroxyl radical footprinting was employed to analyze the interaction of distamycin and actinomycin with the 5s ribosomal RNA genes of Xenopus (Churchill et al, 1990). The two drugs showed different hydroxyl radical footprints. Distamycin gives a conventional (albeit high-resolution) footprint, while actinomycin does not protect DNA from hydroxyl radical attack, but instead induces... 

Chaires JB, Herrera JE, Waring MJ (1990) Preferential binding of daunomycin to 5-ATCG and 5-ATGC sequences revealed footprinting, titration experiment. Biochemistry 29 6145-6153 Chakrabarti S, Mahmood A, Kassis AI, Bump EA, Jones AG, Makrigiorgos GM (1996) Generation of hydroxyl radicals by nucleohistone-bound metal-adriamycin complexes. Free Radic Res... 

The second major area of interaction is near the dyad axis. Hydroxyl radical footprinting revealed protection at 65 and 75 bp from the DNA ends [40]. On the... 

Figure 12-28 (A) A hairpin ribozyme formed from the minus strand of a satellite RNA associated with tobacco ringspot virus. On the basis of hydroxyl radical footprinting (see Fig. 5-50), to identify protected areas a folding pattern that brings domains A and B together to form a compact catalytic core has been proposed.798 (B) A "leadzyme," a ribozyme dependent upon Pb2+ for cleavage of RNA.802 803 (C) An RNA-cleaving DNA enzyme.804...

As the present book is concerned with the free-radical chemistry of DNA, it is worth mentioning that hydroxyl radical footprinting, based on the forma-... 

Tullius TD (1991) DNA footprinting with the hydroxyl radical. Free Radical Res Commun... 

As shown in Fig. 10.3C, comparison with hydroxyl radical footprinting data on the same RNA, under identical experimental condition, revealed that while both native and nonnative tertiary contacts are formed during the first compaction phase, only native tertiary contact formation drives the ribozyme to its folded structure in the second phase. The slightly larger global dimension observed in 1.5 M Na+ as compared to that in 10 mM Mg2+ indicates the inability of monovalent ions, even at sufficiently high concentrations, to fully compact this RNA to its native shape. 

Sclavi, B., Sullivan, M., et al. (1998). RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science 279(5358), 1940-1943. 

These footprinting analyses, based on enzymic and chemical digestion, are now widely used to define DNA (and RNA) and their complexes with various ligands. Recently active radical probes have been used as footprinting agents in protection assays in a variety of systems (e.g., Tullius and Dombroski, 1986 Chalepakis and Beato, 1989 Hayes and Tullius, 1989 Schickor et al., 1990). Such probes rely on active radical intermediates, most likely hydroxyl radicals, released by Fe(II) in the presence of an electron donor, probably via a Fenton reaction. In addition, hydroxyl radicals also appear to react with DNA in a conformation-specific manner which may allow some prediction of DNA secondary structure (see Burkhoft and Tullius, 1987 Zorbas et al., 1989 Lu et al., 1990). 

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