Probing tRNA interaction with biogenic polyamines

Abstract

Biogenic polyamines are found to modulate protein synthesis at different levels. This effect may be explained by the ability of polyamines to bind and influence the secondary structure of tRNA, mRNA, and rRNA. We report the interaction between tRNA and the three biogenic polyamines putrescine, spermidine, spermine, and cobalt(III)hexamine at physiological conditions, using FTIR spectroscopy, capillary electrophoresis, and molecular modeling. The results indicated that tRNA was stabilized at low biogenic polyamine concentration, as a consequence of polyamine interaction with the backbone phosphate group. The main tRNA reactive sites for biogenic polyamine at low concentration were guanine-N7/O6, uracil-O2/O4, adenine-N3, and 2'OH of the ribose. At high polyamine concentration, the interaction involves guanine-N7/O6, adenine-N7, uracil-O2 reactive sites, and the backbone phosphate group. The participation of the polycation primary amino group, in the interaction and the presence of the hydrophobic contact, are also shown. The binding affinity of biogenic polyamine to tRNA molecule was in the order of spermine > spermidine > putrescine with K(Spm) = 8.7 × 10(5) M(-1), K(Spd) = 6.1 × 10(5) M(-1), and K(Put) = 1.0 × 10(5) M(-1), which correlates with their positively charged amino group content. Hill analysis showed positive cooperativity for the biogenic polyamines and negative cooperativity for cobalt-hexamine. Cobalt(III)hexamine contains high- and low-affinity sites in tRNA with K(1) = 3.2 × 10(5) M(-1) and K(2) = 1.7 × 10(5) M(-1), that have been attributed to the interactions with guanine-N7 sites and the backbone PO(2) group, respectively. This mechanism of tRNA binding could explain the condensation phenomenon observed at high Co(III) content, as previously shown in the Co(III)-DNA complexes.

STRUCTURE 1.

Chemical structures of biogenic polyamines putrescine, spermidine, and spermine.

FIGURE 1.

FTIR spectra in the region of 1800–600 cm−1 for pure tRNA, free polyamine, spermine–tRNA (A), and spermidine–tRNA (B) adducts in aqueous solution at pH 7.0 ± 0.2 (top three spectra) and difference spectra for polyamine–tRNA adducts obtained at various polyamine/tRNA(P) molar ratios (bottom two spectra).

FIGURE 2.

FTIR spectra in the region of 1800–600 cm⁻¹ for pure tRNA, free polyamine, putrescine–tRNA (A), and cobalt(III)hexamine–tRNA (B) adducts in aqueous solution at pH 7.0 ± 0.2 (top three spectra) and difference spectra for polyamine–tRNA adducts obtained at various polyamine/tRNA(P) molar ratios (bottom two spectra).

FIGURE 3.

Relative intensity variations in arbitrary units for several tRNA in-plane vibrations as a function of polyamine concentration. (A–D) Relative intensity for the tRNA bands at 1698 (guanine), 1653 (uracil), 1608 (adenine), 1488 (cytosine), and 1244 cm⁻¹ (PO₂⁻) for spermine, spermidine, putrescine, and cobalt(II1I)hexamine, respectively.

FIGURE 4.

(A) Plot of the difference in migration time (in minutes) of polyamine–tRNA complexes from capillary electrophoresis following incubation of a constant concentration of tRNA (1.25 mM) with various concentrations of polyamines. The difference in migration time of the polyamine–tRNA complexes was determined by subtracting the migration time of pure tRNA from that of each polyamine–tRNA adducts. (B,C) Scatchard plots for biogenic polyamine–tRNA, and cobalt(III)hexamine–tRNA complexes, respectively. (D) Hill plots for polyamine–tRNA complexes.

FIGURE 5.

Best conformations for polyamines docked to tRNA (PDB entry 6TNA). (Green) The polyamines. (A) tRNA in sphere-filling model with the putrescine binding site in sticks; (A′) putrescine binding sites represented in sticks with the corresponding base residues. (B) tRNA in sphere-filling model with the spermidine binding site in sticks; (B′) spermidine binding sites represented in sticks. (C) tRNA in sphere-filling model with the spermine binding sites represented in sticks; (C′) the binding sites represented in sticks.