Nonintercalative binding of ethidium bromide to nucleic acids: crystal structure of an ethidium--tRNA molecular complex.

Abstract

X-ray diffraction studies at 4.5A resolution on crystals of a complex of ethidium bromide and yeast phenylalanine tRNA reveal a nonintercalative mode of binding of the ethidium within the tertiary structure. This is contrary to the expected intercalative binding to the double-helical regions.The nature of the interaction of polycyclic drugs, dyes, and mutagens with nucleic acids has been a problem of longstanding interest in the study of the mechanism of drug inhibition of protein and nucleic acid synthesis. Lerman hypothesized that planar drug molecules intercalated between adjacent base pairs of double-helical nucleic acids (1). Solution studies on DNA and RNA have been interpreted to support this hypothesis (2). It has long been assumed that the inhibition of nucleic acid synthesis by the drug ethidium bromide (EtdBr) (Fig. 1) is due to an intercalative mode of binding (1-8). High-resolution nuclear magnetic resonance (NMR) studies have shown that ethidium binds to a single site in yeast phenylalanine tRNA (tRNAPhe) as well as unfractionated tRNAs, and this site has been interpreted as involving intercalation between the amino acid stem base pairs U6-A67 (AU6) and U7-A66 (AU7) (9). Fluorescence (8, 10) and other binding studies (5-7) also implicated an intercalative mode of binding in this region. We have determined the location of the binding site by x-ray diffraction studies of the crystalline molecular complex of EtdBr with yeast tRNAPhe using difference Fourier electron density maps and find a new, unexpected mode of interaction of ethidium within the tRNA tertiary structure that does not involve intercalation (11,12). The ethidium is found to bind specifically and is lodged in a cavity at the mouth of the "P10 loop" of the tRNA tertiary structure. The dye is stacked over U8, which is involved in the invariant tertiary base pair U8-A14, and is hydrogen bonded to the phosphates P8 and P15 across this loop. In light of our observations, we have reinterpreted the NMR spectra (9, 13-16) and have concluded that the interactions of ethidium with tRNA in solution are similar to those in the crystal. The nonintercalative mode of binding within the tertiary structure of tRNA has not been previously conceived and may be important in the function of drug and dye molecules. EXPERIMENTAL Monoclinic crystals of yeast tRNAPhe were grown in 10 mM MgCI2/10 mM cacodylate/1 mM spermine at pH 6.0, using 2-methyl-2,4-pentanediol as the precipitating agent (17). tRNAPhe crystals were soaked in solutions containing a 10-to 20-fold molar excess of EtdBr. The crystals acquired a deep stain in 2-3 days and failure in attempts to wash out the EtdBr indicated the formation of a stable complex. Two independent experiments were performed using crystals before washing (EtdBrl) and after washing (EtdBr2), and they yielded identical results as described below.The unit cell dimensions of the EtdBr-tRNA complex crystals

The phosphate clamp: sequence selective nucleic acid binding profiles and conformational induction of endonuclease inhibition by cationic Triplatin complexes

“…Overall, the ability of Triplatin complexes to displace fluorogenically bound EtBr and stabilize thermal denaturation of tRNA appears favored by shorter linker chain length molecules. It is possible, however, that Triplatin molecules may bind tRNA at non-fluorescent EtBr binding sites ( 41 ) that have negligible influence on thermal denaturation. Atomic force microscopy (AFM) experiments on tRNA adducted by AH78 do indeed show condensation of the ribonucleotide but the results are not inconsistent if there is a subset of Triplatin binding sites different to those of EtBr ( 12 ).…”

Sequence/structure selective thermal and photochemical cleavage of yeast-tRNAPhe by UO22+

“…Such cleavage is interpreted in terms of a high-affinity uranyl binding site (Jeppesen and Nielsen, 1989;Mollegaard et al, 1994), and this by inferrence a highaffinity metal-ion binding site in general, for which Mg2+ is a likely biologically relevant occupant. Therefore, in terms of RNA structure, it is interesting that other cationic probes, such as Pb2+ (Brown er al., 1983) and some rhodium trisphenanthroline complexes (Chow and Barton, 1990) and possibly ethidium bromide (Lienman et al, 1977) and other cationic intercalators (Nielsen,198 1) also seem to recognize the same tertiary interactions. Most notably, Mg2+ ions have also been identified at this position (Fig.…”

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe

“…Unlike the fluorescein case, ethidium bromide binding sites are noncovalent, and their positions and numbers are less well understood (Bittman, 1969;Wells & Cantor, 1977 Liebman et al, 1977). Ethidium bromide, though, does have several advantages.…”

“…It is more environmentally sensitive than fluorescein (and therefore more likely to be influenced by changes in its surroundings), has a longer lifetime than fluorescein (which facilitates polarization studies on large macromolecules such as the ternary complex), and binds to nucleic acids via intercalation or stacking (which results in substantially reduced local mobility compared to covalently attached probes). Although the ethidium binding sites in solution have not been unequivocally determined, crystal studies have placed the single tight binding site at the tRNA "P-10" cavity area (Liebman et al, 1977) near the same s4U8 residue to which the fluorescein probe is covalently attached in tRNAPhe-Fl8. Spectral changes monitored with either ethidium or fluorescein will therefore result from structural changes in the same general region of the tRNA.…”