Inhibition of RNase P RNA cleavage by aminoglycosides

Abstract

A number of aminoglycosides have been reported to interact and interfere with the function of various RNA molecules. Among these are 16S rRNA, the group I intron, and the hammerhead ribozymes. In this report we show that cleavage by RNase P RNA in the absence as well as in the presence of the RNase P protein is inhibited by several aminoglycosides. Among the ones we tested, neomycin B was found to be the strongest inhibitor with a Ki value in the micromolar range (35 microM). Studies of lead(II)-induced cleavage of RNase P RNA suggested that binding of neomycin B interfered with the binding of divalent metal ions to the RNA. Taken together, our findings suggest that aminoglycosides compete with Mg2+ ions for functionally important divalent metal ion binding sites. Thus, RNase P, which is an essential enzyme, is indeed a potential drug target that can be used to develop new drugs by using various aminoglycosides as lead compounds.

Figure 1

Cleavage by M1 RNA alone at 37°C under various conditions as indicated. The final concentrations of reactants and buffer conditions were as outlined in Materials and Methods. The final concentration of neomycin was 1 mM. Lanes 1–3, cleavage in buffer I, where lane 1 = no M1 RNA added, lane 2 = cleavage in the absence of neomycin B, and lane 3 = cleavage in the presence of neomycin B. Time of cleavage for lanes 2 and 3, 0.5 min. Lanes 4–6, cleavage in buffer II, where lane 4 = no M1 RNA added, lane 5 = cleavage in the absence of neomycin B, and lane 6 = cleavage in the presence of neomycin B. Time of cleavage for lanes 5 and 6 = 1 min. Lanes 7–10, cleavage in buffer II at pH values as indicated; lanes 7 and 9 = cleavage in the absence of neomycin and lanes 8 and 10 = cleavage in the presence of neomycin B. Time of cleavage for lanes 7–10 = 3 min.

Figure 2

Cleavage efficiency in percent as a function of increasing concentrations of the various aminoglycosides used in this study. Cleavage was performed at 37°C as outlined in the text. (Bottom) Hill plot analysis (27).

Figure 3

Structures of the two pairs of aminoglycosides used in this study, neomycin B and paromomycin, and kanamycin A and B. Positions where these aminoglycosides differ are indicated with R₁ and the pK_a values for the different ammonium groups are given in the figure.

Figure 4

Cleavage efficiency in percent as a function of increasing concentration of Mg²⁺ in the absence and in the presence of neomycin B. Cleavage was performed in buffer II at 37°C as outlined in the text with a final concentration of neomycin B of 1 mM.

Figure 5

Illustration of a secondary structure model of RNase P RNA derived from E. coli (M1 RNA; ref. 40). The specific Mg²⁺-induced cleavage sites are indicated by arrows and italic roman numerals, while the arrows with nonitalic roman numerals indicate the Pb²⁺-induced cleavage sites. The boxed (shaded box) nucleotides represent residues that are involved in base pairing with the 3′-terminal RCCA sequence of the substrate, the “RCCA-RNase P RNA” interaction (for details see text). The dashed arrow indicates the “P15-loop,” and the M1 RNA derivatives carrying a substitution at position 254 or at position 255 are indicated with open boxes. (Inset) A secondary structure of the tRNA precursor substrate, pSu3, used in this study. The arrow indicate the RNase P cleavage while the boxed residues were deleted in the substrate that lacked the 3′-terminal CCA sequence. The construction and generation of these substrates have been described elsewhere (ref. and references therein).

Figure 6

Lead(II)-induced cleavage of M1 RNA in the presence and in the absence of increasing concentrations of neomycin B or paromomycin as indicated. The final concentrations of aminoglycoside were 10, 100, 500, and 1000 μM. Lanes 5 and 10 = Pb²⁺ cleavage of M1 RNA in the absence of aminoglycoside and lane 11 = no Pb²⁺ or aminoglycoside added. Cleavage was performed as outlined in the text at 37°C and the time of Pb²⁺ cleavage was 10 min in all cases. The roman numerals refer to the positions of cleavage in M1 RNA as shown in Fig. 5.