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

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.

Figure 1.

The polyamine biosynthetic pathway in S. cerevisiae. (A) Antizyme (Oaz1), the main regulator is indicated; it targets ODC (Spe1) for degradation by the 26S proteasome. Metabolites are shown in italics and proteins in Roman type. Asterisks denote genes deleted in the spe1 spe2 paa1 fms1 deletant strain. See text for further details. (B) Block diagram representing the regulation of polyamine biosynthesis by antizyme as a modular feedback control system.

Figure 2.

Antizyme ribosomal frameshifting and readthrough in wild-type BY4741 and spe1 spe2 paa1 fms1 deletant strains. Average percent frameshifting (FS) and readthrough (RT) for wild-type and quadruple deletant strains grown in polyamine-free media. Filled bars represent average frameshift and open bars represent readthrough. Standard deviations are presented for all bars (typically <15% of mean value; n = 3).

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.

Figure 4.

Combinatorial effects of polyamines on frameshifting at the antizyme frameshift site; experimental data and model prediction. Frameshifting frequencies were measured using a dicistronic reporter vector containing the OAZ1 recoding site. Intracellular polyamine concentrations were measured by HPLC. The combined effect of spermidine and spermine (A), putrescine and spermidine (B) and putrescine and spermine (C) on frameshifting are plotted as filled circles, and the model prediction (mesh surface) depicting the fitted frameshift function (Equation 5). Note that panel (C) data points are derived from combined putrescine/spermine treatments some of which contained trace spermidine contamination, but the mesh surface indicates predicted responses to dual spermine/putrescine treatment only. For clarity, experimental data standard deviations are not presented but were typically <15% of mean value (n = 3).

Figure 5.

Validation of the ribosomal controller frameshift model. (A) Polyamine concentrations were measured in a spe2 mutant with and without SPE1 gene overexpression, in an oaz1 deletant, and in the wild-type strain (putrescine; filled bar, spermidine; light grey, spermine; dark grey). Error bars represent standard deviations (n = 3). (B) Ribosomal frameshift frequencies were measured in the same mutant panel (filled bars) and the intracellular polyamine concentrations measured in the mutants were fed into the frameshift function to predict the frameshift frequency (open bars; error bars represent model uncertainty range originating with variation in the experimental data).