Opposite effect of polyamines on In vitro gene expression: Enhancement at low concentrations but inhibition at high concentrations

Abstract

BackgroundPolyamines have various biological functions including marked effects on the structure and function of genomic DNA molecules. Changes in the higher-order structure of DNA caused by polyamines are expected to be closely related to genetic activity. To clarify this issue, we examined the relationship between gene expression and the higher-order structure of DNA under different polyamine concentrations.Principal findingsWe studied the effects of polyamines, spermidine SPD(3+) and spermine SP(4+), on gene expression by a luciferase assay. The results showed that gene expression is increased by ca. 5-fold by the addition of SPD(3+) at 0.3 mM, whereas it is completely inhibited above 2 mM. Similarly, with SP(4+), gene expression is maximized at 0.08 mM and completely inhibited above 0.6 mM. We also performed atomic force microscopy (AFM) observations on DNA under different polyamine concentrations. AFM revealed that a flower-like conformation is generated at polyamine concentrations associated with maximum expression as measured by the luciferase assay. On the other hand, DNA molecules exhibit a folded compact conformation at polyamine concentrations associated with the complete inhibition of expression. Based on these results, we discuss the plausible mechanism of the opposite effect, i.e., enhancement and inhibition, of polyamines on gene expression.Conclusion and significanceIt was found that polyamines exert opposite effect, enhancement and inhibition, on gene expression depending on their concentrations. Such an opposite effect is argued in relation to the conformational change of DNA: enhancement is due to the parallel ordering of DNA segments that is accompanied by a decrease in the negative charge of double-stranded DNA, and inhibition is caused by the compaction of DNA into a tightly packed state with almost perfect charge-neutralization.

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

“…The addition of Na + or K + to the solution tends to increase the luminescence intensity, and K + has a greater enhancing effect, except in experiments with 1.5 mM SPD. The complete inhibition at 1.5 mM SPD is attributable to the transition of the higher-order structure to a tightly packed state, as suggested previously [21,22]. The enhancement of gene expression in the presence of K + has been confirmed at different SPD concentrations as revealed in S1 Fig. To shed light on the effects of Na + and K + on the higher-order structure of DNA in the presence of different concentrations of SPD, we performed real-time single-molecule observation of T4 DNA fluctuating in aqueous solution by using fluorescence microscopy (FM).…”

“…Recently, we have reported that polyamines can either enhance or inhibit gene expression depending on their concentration [21,22]. In our study on the effects of polyamine on gene expression, we observed that, in the presence of polyamine, K + enhances gene expression in vitro much more strongly than Na + .…”

“…order structure of DNA, it has been revealed that individual DNA molecules undergo a discrete transition from an elongated state to a compact folded state with an increase in polyamines, such as SPD and spermine [21,23,29,30]. The observation of the conformation of single DNA molecules, as reflected in the histogram in Fig 3, indicates the coexistence of elongated and compact states for intermediate concentrations of SPD.…”

“…A cell-free in vitro luciferase assay was performed with a TnT T7 Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's instructions and previous reports [21,22]. Plasmid DNA (4331 kbp) was used as the DNA template.…”

“…Allahverdi et al reported that Na + and K + had opposite effects on nucleosome array folding [46] and on the structural integrity of chromosomes [47]. As for effect of polyamine on in viro gene expression, Kanemura et al reported biphasic effect, acceleration and complete inhibition, depending on the concentration of polyamine [21]. The complete inhibition is caused accompanying by the transition of DNA into a compact state at higher concentration of polyamine.…”