Chemical and molecular biology of a eukaryotic riboswitch

生物体的基因组由它的所有基因组成，从细菌的几千个基因到动植物等高等生物的三万个基因不等。这些基因由DNA组成，DNA被转录(复制)成信使RNA(信使RNA)，信使RNA随后被用作模板，将氨基酸组装成蛋白质。即使在最简单的细胞中，也不是所有的基因都能一直表达。为了控制细胞新陈代谢，并对环境变化(如不同营养物质的可获得性)做出反应，细胞必须能够调节其基因的表达。直到最近，这种控制一直被认为完全受蛋白质因素的影响，当蛋白质因素暴露在不同的代谢物中时，能够与DNA或RNA结合，并改变基因表达。在过去的几年里，一种不同的机制被证明是起作用的，通过这种机制，代谢物可以直接与信使核糖核酸相互作用，并通过改变信使核糖核酸的结构，干扰基因的表达。与代谢物结合的mRNA区域被称为核糖开关，因为它们充当代谢开关，开启或关闭基因表达。大多数关于核糖开关的研究都集中在细菌上，细菌的DNA在细胞质中是游离的。人们对真核细胞中的这种现象知之甚少，真核细胞中的DNA包含在细胞核中。我们已经在莱茵衣藻中发现了核糖开关的证据，这是一种真核绿藻--一种简单的植物。像所有的植物一样，衣藻需要制造它所有的氨基酸，其中一种叫做蛋氨酸。衣藻有两种酶来制造蛋氨酸：冰毒和冰毒。冰毒本身可以起作用，但冰毒需要Vitmain B12作为辅因子。然而，衣藻不能制造维生素B12，它必须从生长的介质中吸收维生素B12。因此，当缺乏维生素B12时，藻类使用冰毒，但如果存在维生素B12，它可以使用冰毒，这是一种更活跃的酶。我们已经发现，维生素B12会导致mete mRNA消失，而且mete mRNA会与固定在小珠上的维生素B12结合。因此，它很可能受到核糖开关机制的调控。在这项建议中，我们打算使用一些不同的技术来研究维生素B12(钴蛋白)的不同衍生物对mRNA序列不同部分的亲和力，确定mRNA中哪些核苷酸对结合钴胺是重要的，然后对这些核苷酸进行突变，看看这是否会减少代谢物结合。我们将通过监测mRNA的水平，并将核糖开关与一种名为荧光素酶的酶的基因联系起来，来测试这些突变在细胞中的影响。荧光素酶的表达可以通过它发光的事实来监测。我们还将研究钴胺-信使核糖核酸复合体的结构。然后我们将确定这些核糖开关在细胞中的实际功能。核糖开关可以干扰许多不同的过程，如转录、信使核糖核酸的加工或翻译，以及RNA的降解。我们将进行几个实验来确定哪些过程是由核糖开关控制的。在这些实验中，我们将使用天然的核糖开关序列，以及我们将在提案的第一部分中创建的突变序列。最后，我们将研究衣藻属中的另外两个基因也受核糖开关机制调控的可能性。

The genome of an organism is composed of all of its genes, which can range from a few thousand in bacteria to 30,000 in higher organisms such as plants and animals. The genes are made of DNA, which is transcribed (copied) into messenger RNA (mRNA), which is then used as a template to assemble amino acids into proteins. Even in the simplest cells not all of the genes are expressed all of the time. In order to control cellular metabolism, and respond to changes in the environment (such as the availability of different nutrients), a cell must be able to regulate the expression of its genes. Until recently, this control was thought to be exclusively under the influence of protein factors, which, when exposed to different metabolites, are able to bind DNA or RNA, and alter gene expression. In the last few years a different mechanism has been shown to operate, whereby a metabolite can directly interact with mRNA and, by changing the structure of the mRNA, interfere with the expression of the gene. The regions of mRNA that bind the metabolites have been called riboswitches, because they act as metabolic switches, turning gene expression on or off. Most of the research that has been carried out on riboswitches has focused on bacteria, whose DNA is free in the cytoplasm of the cell. Much less is known about the phenomenon in eukaryotes, cells where the DNA is contained in the nucleus. We have found evidence for a riboswitch in Chlamydomonas reinhardtii, which is a eukaryotic green alga - a simple plant. Like all plants, Chlamydomonas needs to make all its amino acids, one of which is called methionine. Chlamydomonas has two enzymes to make methionine: MetE and MetH. MetE can work on its own, but MetH needs vitmain B12 as a cofactor. However, Chlamydomonas cannot make vitamin B12, it has to take it up from the medium in which it grows. So when vitamin B12 is absent, the alga uses MetE, but if vitamin B12 is present it can use MetH, which is a more active enzyme. We have found that vitamin B12 causes the metE mRNA to disappear, and furthermore the metE mRNA binds to vitamin B12 immobilised on small beads. It is therefore very likely to be regulated by a riboswitch mechanism. In this proposal we intend to use a number of different techniques to investigate the affinity of different derivatives of vitamin B12 (cobalamins) for different parts of the mRNA sequence, determine which nucleotides within the mRNA are important for binding cobalamin, and then mutate these to see if this diminishes metabolite binding. We will test the effect of these mutations in the cell by monitoring the levels of mRNA, and by linking the riboswitches to the gene for an enzyme called luciferase. The expression of luciferase can be monitored by the fact that it gives off light. We will also investigate the structure of the cobalamin-mRNA complex. We will then determine how these riboswitches actually function in the cell. Riboswitches could interfere with a number of different processes, such as transcription, mRNA processing or translation, as well as the degradation of RNA. We will carry out several experiments to determine which processes are controlled by riboswitches. In these experiments we will use the natural riboswitch sequence, as well as the mutant sequences that we will have created in the first part of the proposal. Finally, we will investigate the possibility that two other genes in Chlamydomonas are also regulated by a riboswitch mechanism.