K+ promotes the favorable effect of polyamine on gene expression better than Na

Abstract

Background: Polyamines are involved in a wide variety of biological processes including a marked effect on the structure and function of DNA. During our study on the interaction of polyamines with DNA, we found that K+ enhanced in vitro gene expression in the presence of polyamine more strongly than Na+. Thus, we sought to clarify the physico-chemical mechanism underlying this marked difference between the effects of K+ and Na+.

Principal findings: It was found that K+ enhanced gene expression in the presence of spermidine, SPD(3+), much more strongly than Na+, through in vitro experiments with a Luciferase assay on cell extracts. Single-DNA observation by fluorescence microscopy showed that Na+ prevents the folding transition of DNA into a compact state more strongly than K+. 1H NMR measurement revealed that Na+ inhibits the binding of SPD to DNA more strongly than K+. Thus, SPD binds to DNA more favorably in K+-rich medium than in Na+-rich medium, which leads to favorable conditions for RNA polymerase to access DNA by decreasing the negative charge.

Conclusion and significance: We found that Na+ and K+ exhibit markedly different effects through competitive binding with a cationic polyamine, SPD, to DNA, which causes a large difference in the higher-order structure of genomic DNA. It is concluded that the larger favorable effect of Na+ than K+ on in vitro gene expression observed in this study is well attributable to the significant difference between Na+ and K+ on the competitive binding inducing conformational transition of DNA.

Fig 1

Efficiency of gene expression vs. concentrations of (A) SPD, (B) Na⁺ and (C) K⁺. C⁰ is the concentration of Na⁺ and K⁺ contained in the original reaction buffer; C = C⁰+ΔC. The intensity is normalized to the control condition (= 1), where ΔC = 0 in the absence of SPD. The DNA concentration was fixed at 0.3 μM.

Fig 2. Examples of FM images of a single T4 DNA molecule undergoing Brownian motion in solution.

(A) In the absence of any condensation agent such as SPD, Na⁺ or K⁺. (B) In the presence of 0.3 mM SPD. (C) In the presence of 0.3 mM SPD and 30 mM ΔC_Na. The total observation time for (A)–(C) is 3 s.

Fig 3. Histograms for the long-axis length L of T4 DNA at different concentrations of C_Na or C_K with 0–1.5 mM SPD.

Fig 4. Evaluation of the binding affinity of Na⁺ and K⁺ for DNA through ¹H NMR measurements.

(A) ¹H NMR signals of 0.1 mM SPD in D₂O solution. (B) ¹H NMR signals of 0.1 mM SPD with different concentrations of Na⁺ or K⁺ in the presence of 1.6 mM CT DNA. (C) Changes in the integrated intensity of ¹H NMR signals, I_NMR, depending on the concentrations C. The intensities in the graph were evaluated from the sum of all integrated values for the signals of SPD. (D) Log-log plot; proportion of unbound SPD to bound SPD, P_Free/P_Bound, vs. the salt concentrations C. P_Free and P_Bound are evaluated from the relationship; P_Free = I_NMR and P_Bound = 1 –I_NMR, respectively.