Control of a Plant Translational Regulatory Protein Through Nutrient Signaling

烟草花叶病毒RNA的5'先导体（称为ω）起翻译增强子的作用。对一个102 kDa蛋白的序列分析显示，该蛋白与HSP101家族同源，其在酵母中的表达弥补了由HSP104基因缺失引起的耐热性缺陷。在表达烟草HSP101的酵母中，omega-luc mRNA的翻译量增加了20倍，而在没有HSP101的情况下，omega没有增强翻译。因此，HSP101和omega组成了一个双组分的翻译调控机制，可以在酵母中重现。对酵母翻译突变体中HSP101功能的分析表明，HSP101介导的增强需要启动因子eIF3和eIF4G。HSP101的rna结合和翻译调控活性在呼吸细胞、氨基酸饥饿或指数后期/平稳生长过程中不活跃，而其耐热功能不受影响。这导致了一个模型，在这个模型中，HSP101作为翻译调节剂的功能需要激活其rna结合活性，而rna结合活性反过来又由营养信号控制。该项目的第一个目标是通过缺失和突变分析确定rna结合位点，研究HSP101作为rna结合蛋白的功能。第二个目标是确定它的rna结合活性是如何受到营养供应的调节的。本研究旨在了解一种受热休克诱导的蛋白（HSP101）如何调控烟草花叶病毒蛋白的合成。由于负责这一调控的两个组成部分，称为omega的RNA序列和烟草HSP101蛋白，在酵母中表达时足以概括这一调控，因此该项目将研究HSP101在与RNA结合和调节酵母翻译中的作用。该项目将进一步研究酵母中HSP101功能通过营养可利用性的调节。因此，这项研究将增加我们对赋予植物调节蛋白质生产能力的机制的理解。

Gallie The 5'-leader (called omega) of tobacco mosaic viral RNA functions as a translational enhancer. Sequence analysis of a 102 kDa protein, previously identified as a specific omega RNA-binding protein, revealed homology to the HSP101 family and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 20-fold increase in the translation of omega-luc but not luc mRNA was observed in yeast expressing the tobacco HSP101 whereas omega failed to enhance translation in the absence of HSP101. Therefore, HSP101 and omega comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factors eIF3 and eIF4G were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells, following amino acid starvation, or during late-exponential/stationary growth, whereas its thermotolerance function remained unaffected. This has led to a model in which the function of HSP101 as a translational regulator requires activation of its RNA-binding activity which in turn is controlled by nutrient signaling. The first goal of this project is to investigate how HSP101 functions as an RNA-binding protein by identifying the RNA-binding site through deletion and mutation analysis. The second goal will determine how its RNA-binding activity is regulated by nutrient availability. This research seeks to understand how a protein that is induced in response to heat shock (HSP101) regulates the synthesis of tobacco mosaic virus proteins. Since the two components that are responsible for this regulation, an RNA sequence called omega and the tobacco HSP101 protein, are sufficient to recapitulate this regulation when expressed in yeast, this project will examine the role of HSP101 in binding to RNA and regulating translation in yeast. The project will further examine the regulation of HSP101 function in yeast by nutrient availability. Thus, this research will increase our understanding of a mechanism that confers on plants the ability to regulate protein production.