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Translational recoding as a feedback controller: systems approaches reveal polyamine-specific effects on the antizyme ribosomal frameshift.

Rato C, Amirova SR, Bates DG, Stansfield I, Wallace HM - Nucleic Acids Res. (2011)

Bottom Line: Combinatorial polyamine treatments showed polyamines compete for binding to common ribosome sites.Using concepts from enzyme kinetics and control engineering, a mathematical model of the translational controller was developed to describe these complex ribosomal responses to combinatorial polyamine effects.Each one of a range of model predictions was successfully validated against experimental frameshift frequencies measured in S-adenosylmethionine-decarboxylase and antizyme mutants, as well as in the wild-type genetic background.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, UK.

ABSTRACT
The antizyme protein, Oaz1, regulates synthesis of the polyamines putrescine, spermidine and spermine by controlling stability of the polyamine biosynthetic enzyme, ornithine decarboxylase. Antizyme mRNA translation depends upon a polyamine-stimulated +1 ribosomal frameshift, forming a complex negative feedback system in which the translational frameshifting event may be viewed in engineering terms as a feedback controller for intracellular polyamine concentrations. In this article, we present the first systems level study of the characteristics of this feedback controller, using an integrated experimental and modeling approach. Quantitative analysis of mutant yeast strains in which polyamine synthesis and interconversion were blocked revealed marked variations in frameshift responses to the different polyamines. Putrescine and spermine, but not spermidine, showed evidence of co-operative stimulation of frameshifting and the existence of multiple ribosome binding sites. Combinatorial polyamine treatments showed polyamines compete for binding to common ribosome sites. Using concepts from enzyme kinetics and control engineering, a mathematical model of the translational controller was developed to describe these complex ribosomal responses to combinatorial polyamine effects. Each one of a range of model predictions was successfully validated against experimental frameshift frequencies measured in S-adenosylmethionine-decarboxylase and antizyme mutants, as well as in the wild-type genetic background.

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Single treatment effects of putrescine, spermidine and spermine on frameshifting at the antizyme frameshift site. Average percent frameshifting (filled circles) was measured using a dicistronic assay and plotted versus intracellular polyamine intracellular concentrations in the spe1 spe2 paa1 fms1 deletant strain. (A) Putrescine effects on frameshifting. The highest putrescine concentration used contained 0.16 mM contaminating spermidine, and was therefore not used in the curve fitting process. (B) Putrescine-stimulated frameshift frequencies were measured in the quadruple deletant strain transformed with pGAL1-SPE1 grown on a range of galactose concentrations to regulate SPE1 expression. (C) The effect of intracellular spermidine on frameshift frequency. (D) The effect of intracellular spermine on frameshift frequency. The highest concentration contained 0.2 mM contaminating spermidine. For reference, the wild-type strain BY4741 frameshift frequency (open circles) is represented on all graphs. Error bars (horizontal and vertical) indicate standard deviations for three independent transformants, analysed in triplicate.
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Figure 3: Single treatment effects of putrescine, spermidine and spermine on frameshifting at the antizyme frameshift site. Average percent frameshifting (filled circles) was measured using a dicistronic assay and plotted versus intracellular polyamine intracellular concentrations in the spe1 spe2 paa1 fms1 deletant strain. (A) Putrescine effects on frameshifting. The highest putrescine concentration used contained 0.16 mM contaminating spermidine, and was therefore not used in the curve fitting process. (B) Putrescine-stimulated frameshift frequencies were measured in the quadruple deletant strain transformed with pGAL1-SPE1 grown on a range of galactose concentrations to regulate SPE1 expression. (C) The effect of intracellular spermidine on frameshift frequency. (D) The effect of intracellular spermine on frameshift frequency. The highest concentration contained 0.2 mM contaminating spermidine. For reference, the wild-type strain BY4741 frameshift frequency (open circles) is represented on all graphs. Error bars (horizontal and vertical) indicate standard deviations for three independent transformants, analysed in triplicate.

Mentions: The effect of polyamines on frameshifting at the OAZ1 frameshift site was next examined in the quadruple deletant strain grown in media with varying extracellular polyamine concentrations. The addition of increasing extracellular concentrations of putrescine (10−1, 1, and 10 mM) produced either no effect, or a very minor 1.7-fold stimulatory effect on frameshift frequencies (Figure 3A). However, other studies have shown that very high concentrations of putrescine can stimulate frameshifting at the yeast Ty1 retrotransposon frameshift site, specifically under conditions where very low concentrations of spermidine and spermine were present (35). We therefore overexpressed the SPE1 gene under the control of the GAL1 promoter to engineer higher intracellular concentrations of putrescine. Using galactose in the culture medium to induce SPE1, extremely high levels of putrescine (18.6–71 mM) were attained. At these very high-intracellular concentrations, frameshifting was indeed stimulated, to a maximum of 34% (Figure 3B). Strikingly, the putrescine frameshift response curve was sigmoidal, indicating that the binding of putrescine to the ribosome to stimulate frameshifting was in some way cooperative and may involve multiple binding sites (Figure 3B).Figure 3.


Translational recoding as a feedback controller: systems approaches reveal polyamine-specific effects on the antizyme ribosomal frameshift.

Rato C, Amirova SR, Bates DG, Stansfield I, Wallace HM - Nucleic Acids Res. (2011)

Single treatment effects of putrescine, spermidine and spermine on frameshifting at the antizyme frameshift site. Average percent frameshifting (filled circles) was measured using a dicistronic assay and plotted versus intracellular polyamine intracellular concentrations in the spe1 spe2 paa1 fms1 deletant strain. (A) Putrescine effects on frameshifting. The highest putrescine concentration used contained 0.16 mM contaminating spermidine, and was therefore not used in the curve fitting process. (B) Putrescine-stimulated frameshift frequencies were measured in the quadruple deletant strain transformed with pGAL1-SPE1 grown on a range of galactose concentrations to regulate SPE1 expression. (C) The effect of intracellular spermidine on frameshift frequency. (D) The effect of intracellular spermine on frameshift frequency. The highest concentration contained 0.2 mM contaminating spermidine. For reference, the wild-type strain BY4741 frameshift frequency (open circles) is represented on all graphs. Error bars (horizontal and vertical) indicate standard deviations for three independent transformants, analysed in triplicate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113565&req=5

Figure 3: Single treatment effects of putrescine, spermidine and spermine on frameshifting at the antizyme frameshift site. Average percent frameshifting (filled circles) was measured using a dicistronic assay and plotted versus intracellular polyamine intracellular concentrations in the spe1 spe2 paa1 fms1 deletant strain. (A) Putrescine effects on frameshifting. The highest putrescine concentration used contained 0.16 mM contaminating spermidine, and was therefore not used in the curve fitting process. (B) Putrescine-stimulated frameshift frequencies were measured in the quadruple deletant strain transformed with pGAL1-SPE1 grown on a range of galactose concentrations to regulate SPE1 expression. (C) The effect of intracellular spermidine on frameshift frequency. (D) The effect of intracellular spermine on frameshift frequency. The highest concentration contained 0.2 mM contaminating spermidine. For reference, the wild-type strain BY4741 frameshift frequency (open circles) is represented on all graphs. Error bars (horizontal and vertical) indicate standard deviations for three independent transformants, analysed in triplicate.
Mentions: The effect of polyamines on frameshifting at the OAZ1 frameshift site was next examined in the quadruple deletant strain grown in media with varying extracellular polyamine concentrations. The addition of increasing extracellular concentrations of putrescine (10−1, 1, and 10 mM) produced either no effect, or a very minor 1.7-fold stimulatory effect on frameshift frequencies (Figure 3A). However, other studies have shown that very high concentrations of putrescine can stimulate frameshifting at the yeast Ty1 retrotransposon frameshift site, specifically under conditions where very low concentrations of spermidine and spermine were present (35). We therefore overexpressed the SPE1 gene under the control of the GAL1 promoter to engineer higher intracellular concentrations of putrescine. Using galactose in the culture medium to induce SPE1, extremely high levels of putrescine (18.6–71 mM) were attained. At these very high-intracellular concentrations, frameshifting was indeed stimulated, to a maximum of 34% (Figure 3B). Strikingly, the putrescine frameshift response curve was sigmoidal, indicating that the binding of putrescine to the ribosome to stimulate frameshifting was in some way cooperative and may involve multiple binding sites (Figure 3B).Figure 3.

Bottom Line: Combinatorial polyamine treatments showed polyamines compete for binding to common ribosome sites.Using concepts from enzyme kinetics and control engineering, a mathematical model of the translational controller was developed to describe these complex ribosomal responses to combinatorial polyamine effects.Each one of a range of model predictions was successfully validated against experimental frameshift frequencies measured in S-adenosylmethionine-decarboxylase and antizyme mutants, as well as in the wild-type genetic background.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, UK.

ABSTRACT
The antizyme protein, Oaz1, regulates synthesis of the polyamines putrescine, spermidine and spermine by controlling stability of the polyamine biosynthetic enzyme, ornithine decarboxylase. Antizyme mRNA translation depends upon a polyamine-stimulated +1 ribosomal frameshift, forming a complex negative feedback system in which the translational frameshifting event may be viewed in engineering terms as a feedback controller for intracellular polyamine concentrations. In this article, we present the first systems level study of the characteristics of this feedback controller, using an integrated experimental and modeling approach. Quantitative analysis of mutant yeast strains in which polyamine synthesis and interconversion were blocked revealed marked variations in frameshift responses to the different polyamines. Putrescine and spermine, but not spermidine, showed evidence of co-operative stimulation of frameshifting and the existence of multiple ribosome binding sites. Combinatorial polyamine treatments showed polyamines compete for binding to common ribosome sites. Using concepts from enzyme kinetics and control engineering, a mathematical model of the translational controller was developed to describe these complex ribosomal responses to combinatorial polyamine effects. Each one of a range of model predictions was successfully validated against experimental frameshift frequencies measured in S-adenosylmethionine-decarboxylase and antizyme mutants, as well as in the wild-type genetic background.

Show MeSH
Related in: MedlinePlus