Analysing GCN4 translational control in yeast by stochastic chemical kinetics modelling and simulation

BMC Systems Biology, Aug 2011

Background The yeast Saccharomyces cerevisiae responds to amino acid starvation by inducing the transcription factor Gcn4. This is mainly mediated via a translational control mechanism dependent upon the translation initiation eIF2·GTP·Met-tRNAiMet ternary complex, and the four short upstream open reading frames (uORFs) in its 5' mRNA leader. These uORFs act to attenuate GCN4 mRNA translation under normal conditions. During amino acid starvation, levels of ternary complex are reduced. This overcomes the GCN4 translation attenuation effect via a scanning/reinitiation control mechanism dependent upon uORF spacing. Results Using published experimental data, we have developed and validated a probabilistic formulation of GCN4 translation using the Chemical Master Equation (Model 1). Model 1 explains GCN4 translation's nonlinear dependency upon uORF placements, and predicts that an as yet unidentified factor, which was proposed to regulate GCN4 translation under some conditions, only has pronounced effects upon GCN4 translation when intercistronic distances are unnaturally short. A simpler Model 2 that does not include this unidentified factor could well represent the regulation of a natural GCN4 mRNA. Using parameter values optimised for this algebraic Model 2, we performed stochastic simulations by Gillespie algorithm to investigate the distribution of ribosomes in different sections of GCN4 mRNA under distinct conditions. Our simulations demonstrated that ribosomal loading in the 5'-untranslated region is mainly determined by the ratio between the rates of 5'-initiation and ribosome scanning, but was not significantly affected by rate of ternary complex binding. Importantly, the translation rate for codons starved of cognate tRNAs is predicted to be the most significant contributor to the changes in ribosomal loading in the coding region under repressing and derepressing conditions. Conclusions Our integrated probabilistic Models 1 and 2 explained GCN4 translation and helped to elucidate the role of a yet unidentified factor. The ensuing stochastic simulations evaluated different factors that may impact on the translation of GCN4 mRNA, and integrated translation status with ribosomal density.

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Analysing GCN4 translational control in yeast by stochastic chemical kinetics modelling and simulation

BMC Systems Biology Analysing GCN4 translational control in yeast by stochastic chemical kinetics modelling and simulation Tao You 0 Ian Stansfield M Carmen Romano 0 Alistair JP Brown George M Coghill 0 0 School of Natural and Computing Sciences, University of Aberdeen, Institute of Complex System and Mathematical Biology, Aberdeen , UK Background: The yeast Saccharomyces cerevisiae responds to amino acid starvation by inducing the transcription factor Gcn4. This is mainly mediated via a translational control mechanism dependent upon the translation initiation eIF2GTPMet-tRNAiMet ternary complex, and the four short upstream open reading frames (uORFs) in its 5' mRNA leader. These uORFs act to attenuate GCN4 mRNA translation under normal conditions. During amino acid starvation, levels of ternary complex are reduced. This overcomes the GCN4 translation attenuation effect via a scanning/reinitiation control mechanism dependent upon uORF spacing. Results: Using published experimental data, we have developed and validated a probabilistic formulation of GCN4 translation using the Chemical Master Equation (Model 1). Model 1 explains GCN4 translation's nonlinear dependency upon uORF placements, and predicts that an as yet unidentified factor, which was proposed to regulate GCN4 translation under some conditions, only has pronounced effects upon GCN4 translation when intercistronic distances are unnaturally short. A simpler Model 2 that does not include this unidentified factor could well represent the regulation of a natural GCN4 mRNA. Using parameter values optimised for this algebraic Model 2, we performed stochastic simulations by Gillespie algorithm to investigate the distribution of ribosomes in different sections of GCN4 mRNA under distinct conditions. Our simulations demonstrated that ribosomal loading in the 5'-untranslated region is mainly determined by the ratio between the rates of 5'-initiation and ribosome scanning, but was not significantly affected by rate of ternary complex binding. Importantly, the translation rate for codons starved of cognate tRNAs is predicted to be the most significant contributor to the changes in ribosomal loading in the coding region under repressing and derepressing conditions. Conclusions: Our integrated probabilistic Models 1 and 2 explained GCN4 translation and helped to elucidate the role of a yet unidentified factor. The ensuing stochastic simulations evaluated different factors that may impact on the translation of GCN4 mRNA, and integrated translation status with ribosomal density. mRNA translation; GCN4; Gillespie algorithm; stochastic model - Background Reprogramming gene expression is an important means for cells to adapt to environmental changes. In eukaryotes, gene expression is regulated at multiple levels, including transcription, RNA splicing and translation. Translational control mechanisms, particularly acting at the level of translation initiation, can be a primary point of regulation for certain genes. The yeast GCN4 gene is one such example. It encodes a transcription factor that regulates expression of genes encoding amino acid biosynthetic (and other) enzymes. As such, it plays a central role in the amino acid starvation or GCN response [1,2]. GCN4 mRNA translation is regulated through an unusually long 5-leader region (591 nucleotides in length), which contains four short upstream open reading frames (uORFs) (Figure 1A) [2]. While uORFs in 5 leaders can frequently attenuate translation of the downstream open GCN4 mRNA Figure 1 GCN4 translational control. The positions of the uORFs (open boxes) in the 5 leader sequence are drawn roughly to scale. The main GCN4 ORF is depicted partially. Point mutations that remove the start codon of a uORFs are labelled by cross. (A) Wild type GCN4 mRNA structure. (B) Cartoon of GCN4 mRNA translation. (C-E) GCN4-lacZ constructs that were used to investigate GCN4 translation under repressing and derepressing conditions, the activities of which are denoted as A1, A2 and A3, respectively. reading frame, some allow ribosomes to resume scanning following uORF translation. This is dependent on the nature of a roughly 15-nucleotide long sequence immediately downstream of the uORF stop codon [2]. GCN4 uORF1 and uORF2 have this property, and are relatively weak barriers that allow nearly half of the ribosomes to remain on the GCN4 mRNA after their translation, while uORF3 and uORF4 are more inhibitory, causing nearly all of the ribosomes to disassociate from the GCN4 mRNA after their translation [2]. A recent study further suggests that after uORF1 translation, the ribosome dissociation from the mRNA is prevented by a mechanism involving eIF3 interaction with the mRNA [3]. At the beginning of GCN4 mRNA translation, a 43S ribosomal subunit, incorporating an eIF2GTPMettRNAiMet ternary complex (TC), scans from the 5 end of the mRNA to initiate translation at uORF1. Following uORF1 translation termination, about half of (...truncated)


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Tao You, Ian Stansfield, M Carmen Romano, Alistair JP Brown, George M Coghill. Analysing GCN4 translational control in yeast by stochastic chemical kinetics modelling and simulation, BMC Systems Biology, 2011, pp. 131, 5, DOI: 10.1186/1752-0509-5-131