Dynamics of Nitrogen-regulated Gene Expression Reveals a Reciprocal Relationship between Cell Growth Rate and Nitrogen Catabolism

Cell growth rate is regulated in response to resource availability including the abundance, and molecular form, of essential nutrients. In the model eukaryotic cell, Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen impacts both cell growth rate and mRNA expression. Disentangling causal relationships between nitrogen availability, cell growth rate and differential gene expression poses a considerable challenge. Using experimental control of cell growth rate using chemostats, we studied the effect of variation in environmental nitrogen on differential gene expression. We find that the primary determinant of nitrogen-regulated gene expression is nitrogen abundance whereas variation in nitrogen source affects the expression of only a small number of transcripts with highly specialized functions. To study the dynamics of nitrogen-responsive gene expression we perturbed steady-state nitrogen-limited chemostat cultures by addition of either proline or glutamine. Addition of either proline or glutamine to cells growing in nitrogen-limited chemostats results in repression of the nitrogen catabolite repression (NCR) regulon consistent with nitrogen abundance, and not nitrogen source, being the primary determinant of nitrogen-regulated gene expression. We find that a transition from nitrogen-limited to nitrogen-replete conditions is accompanied by rapid induction of transcripts required for protein translation. We identified a reciprocal relationship between specific regulons required for protein translation (RP and RiBi) and the NCR regulon. Using mathematical modeling we find evidence that cells adopt a metabolically inefficient growth mode during this transition. By means of high resolution time series analysis we find evidence that rapid, and potentially accelerated, mRNA degradation plays an important role in remodeling gene expression programs in response to change in environmental nitrogen. We propose that the evolutionarily conserved TORC1 signaling pathway orchestrates the balance between protein translation and assimilation of nitrogen sources at the transcriptional level to optimize rates of cell proliferation. A total of of 102 samples were analyzed in different nitrogen-limited conditions using chemostats in both steady-state and dynamic conditions. A common reference obtained from an ammonium-limited chemostat growing at a dilution rate of 0.12/hr was used for all two color hybridization experiments.

细胞生长速率会响应资源可获得性（包括必需营养素的丰度与分子形式）进行调控。在模式真核细胞酿酒酵母（Saccharomyces cerevisiae，又称出芽酵母）中，环境氮的分子形式会同时影响细胞生长速率与信使RNA（mRNA）表达。厘清氮可获得性、细胞生长速率与差异基因表达之间的因果关系，是一项颇具难度的挑战。我们利用恒化器（chemostat）对细胞生长速率进行实验调控，以此探究环境氮的变化对差异基因表达的影响。研究发现，氮调控基因表达的主要决定因素是氮的丰度，而氮源的变化仅会影响少量具有高度特化功能的转录本的表达。
为探究氮响应型基因表达的动态变化，我们通过向稳态氮限制恒化器培养物中添加脯氨酸或谷氨酰胺，对其进行扰动。向氮限制恒化器中培养的细胞添加脯氨酸或谷氨酰胺，会导致氮分解代谢阻遏（nitrogen catabolite repression, NCR）调控子的表达被抑制，这与“氮丰度而非氮源是氮调控基因表达的主要决定因素”的结论一致。我们发现，从氮限制状态向氮充足状态的转变，会伴随蛋白质翻译所需转录本的快速诱导表达。我们鉴定出蛋白质翻译所需的特定调控子——核糖体蛋白（RP, ribosomal protein）与核糖体生物发生（RiBi, ribosome biogenesis）调控子——与NCR调控子之间存在相互调控关系。通过数学建模，我们发现细胞在该转变过程中会采用一种代谢效率较低的生长模式。借助高分辨率时间序列分析，我们发现快速且可能被加速的mRNA降解，在响应环境氮变化的基因表达程序重塑过程中发挥着重要作用。我们提出，进化保守的雷帕霉素靶蛋白复合物1（TORC1, target of rapamycin complex 1）信号通路会在转录层面协调蛋白质翻译与氮源同化之间的平衡，以优化细胞增殖速率。
本研究通过恒化器在稳态与动态条件下，对不同氮限制条件中的共计102份样本进行了分析。所有双色杂交实验均采用来自稀释速率为0.12/小时的铵限制恒化器培养物制备的通用参考样本。