Abstract
The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.
Highlights
Polyamines [putrescine (PUT), spermidine (SPD) and spermine (SPM)] are present at millimolar concentrations in both prokaryotic and eukaryotic cells, and play important roles in cell proliferation and viability [1,2,3,4]
The effects of NSPD, SPD, HSPD and APCAD (Fig 1) on growth of the E. coli polyamine requiring mutant MA261 were compared at 32°C, 37°C and 42°C (Fig 2A)
The growth of E. coli MA261 was stimulated greatly by 0.1 mM each of NSPD, SPD and HSPD at almost the same extent, and less effectively by APCAD compared to the absence of triamines at both 32°C and 37°C
Summary
Polyamines [putrescine (PUT), spermidine (SPD) and spermine (SPM)] are present at millimolar concentrations in both prokaryotic and eukaryotic cells, and play important roles in cell proliferation and viability [1,2,3,4]. The mechanism of stimulation of protein synthesis involving genes in the polyamine modulon is as follows: 1) Polyamines stimulate protein synthesis when a Shine-Dalgarno (SD) sequence in the mRNA is distant from the initiation codon AUG; 2) Polyamines stimulate protein synthesis when the inefficient initiation codon UUG or GUG is present in the mRNA; and 3) Polyamines stimulate protein synthesis when the open reading frame in mRNAs contains a termination codon. In this case, polyamines stimulate suppressor tRNA binding or +1 frameshift [7]. For polyamine effects at the level of DNA, it has been reported that polyamines can stimulate B to Z conversion of poly(dG-m5dC)poly(dG-m5dC) in a cell-free systems [13, 14], but far there is no report using intact cells
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