Translational expression control in land plant arginine decarboxylase transcripts

多胺是具有两个或更多氨基的代谢物，存在于生命的三个领域：真核生物、细菌和古生物。Pas在活细胞中的普遍存在本身就意味着它们的存在是势在必行的，虽然人们对它们的生物合成有很好的了解，但缺乏一个统一它们的分子作用的单一概念。在植物中，精氨酸脱羧酶(ADC)催化合成腐胺，腐胺是所有多胺的前体。此前，我们已经发现细菌植物病原体青枯菌将一种转录激活因子样效应子(TALE)蛋白注入宿主植物细胞，该蛋白与植物ADC基因结合并在转录上激活植物ADC基因，从而提高宿主PA水平。TALL蛋白结合在ADC基因上游50bp的基序内，该基序在所有陆地植物物种中都是保守的，被指定为ADC-box。在天然的ADC mRNAs中，转录的ADC-box是5‘UTR的一部分，预计会形成一种发夹结构，阻止核糖体向Aug起始密码子移动。与这一模型一致，ADC-box已被证明在高PA水平下抑制ADC转录产物的翻译，因此ADC-box可能是PA动态平衡的自我调节表达控制系统的一部分。在目前的研究方案中，我们的目标是阐明陆地植物ADC转录本中翻译调控的分子基础。从专题上讲，研究提案分为两部分：1)鉴定ADC转录本5‘UTRs中参与植物模式植物番茄和拟南芥表达调控的序列元件。为此，我们将研究由CRISPR或定点突变产生的ADC突变等位基因。发现在ADC转录的翻译中调节反馈控制的细胞组件。在这里，基于报告的分析将识别影响ADC转录物翻译活性的PA相关蛋白和/或代谢物。这一功能方法将与基于相互作用的策略相补充，以发现与ADC转录本5‘UTRs结合的调控成分。上述研究将揭示ADC转录本和PA动态平衡的翻译调控的复杂性。一旦我们破译了ADC转录本中翻译调节的机械原理，我们就可以使用这个调节电路中识别的PA感觉模块来设计基因编码的PA生物传感器。这种生物传感器将使我们能够以高空间和时间分辨率监测体内PA水平的变化，从而为揭示PA调控的生物过程提供关键工具。

Polyamines (PAs) are metabolites with two or more amino groups that exist throughout all three domains of life: eukaryotes, bacteria, and archaea. The ubiquity of PAs in living cells inherently implies that their existence is imperative, and while their biosynthesis is well understood, a singular concept that unifies their molecular role is lacking. In plants, arginine decarboxylase (ADC) catalyzes the synthesis of putrescine, the precursor of all PAs. Previously, we have found that the bacterial plant pathogen Ralstonia solanacearum injects a transcription-activator-like effector (TALE) protein into host plant cells, which binds to and transcriptionally activates plant ADC genes to boost host PA levels. The TALE protein binds within a 50bp-motif upstream of ADC genes, which is conserved across all land plant species, and was designated as the ADC-box. In native ADC mRNAs, the transcribed ADC-box is part of the 5’UTR and is predicted to form a hairpin structure that inhibits movement of the ribosome towards the AUG start codon. In agreement with this model, the ADC-box has been shown to inhibit translation of ADC transcripts at high PA levels, and stands to reason that the ADC-box could be part of an autoregulatory expressional control system for PA homeostasis. Within the current research proposal we aim to elucidate the molecular basis of translational regulation in land plant ADC transcripts. The research proposal is topically divided into two sections:1.) Identify sequence elements within ADC transcript 5’UTRs that contribute to expressional regulation in the plant model species tomato and Arabidopsis. To do so, we will study ADC mutant alleles generated by either CRISPR or site-directed mutagenesis.2.) Uncover cellular components that mediate feedback control in translation of ADC transcripts. Here, reporter-based assays will identify PA-related proteins and/or metabolites that affect translational activity of ADC transcripts. This functional approach will be complemented with interaction-based strategies to uncover regulatory components that bind to ADC transcript 5’UTRs.The aforementioned studies will bring to light the intricacies of the translational regulation of ADC transcripts and PA homeostasis. Once we have decoded the mechanistic principles of translational regulation in ADC transcripts, we can use the identified PA-sensory modules of this regulatory circuit to engineer genetically-encoded PA biosensors. Such biosensors will enable us to monitor changes in PA levels in vivo with high spatial and temporal resolution and thus provide a key tool to uncover PA-regulated biological processes.