Chemical and molecular biology of a eukaryotic riboswitch
真核核糖开关的化学和分子生物学
基本信息
- 批准号:BB/D011043/1
- 负责人:
- 金额:$ 26.12万
- 依托单位:
- 依托单位国家:英国
- 项目类别:Research Grant
- 财政年份:2006
- 资助国家:英国
- 起止时间:2006 至 无数据
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
生物体的基因组由其所有基因组成,从细菌中的几千个到植物和动物等高等生物中的30000个不等。这些基因由DNA组成,DNA被转录(复制)成信使RNA (mRNA),然后信使RNA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在每时每刻表达。为了控制细胞代谢,并对环境的变化(如不同营养物质的可用性)做出反应,细胞必须能够调节其基因的表达。直到最近,这种控制被认为完全受蛋白质因子的影响,当暴露于不同的代谢物时,蛋白质因子能够结合DNA或RNA,并改变基因表达。在过去的几年里,一种不同的机制已经被证明起作用,即代谢物可以直接与mRNA相互作用,并通过改变mRNA的结构,干扰基因的表达。结合代谢物的mRNA区域被称为核糖开关,因为它们充当代谢开关,控制基因表达的开启或关闭。大多数关于核糖开关的研究都集中在细菌上,细菌的DNA在细胞的细胞质中是自由的。人们对真核生物(DNA包含在细胞核中的细胞)中的这种现象知之甚少。我们已经在莱茵衣藻(Chlamydomonas reinhardtii)——一种真核绿藻——一种简单的植物中发现了核开关的证据。像所有的植物一样,衣藻需要制造所有的氨基酸,其中一种叫做蛋氨酸。衣藻有两种酶来制造蛋氨酸:MetE和MetH。甲基安非他命可以单独起作用,但甲基安非他命需要维生素B12作为辅助因子。然而,衣藻不能制造维生素B12,它必须从它生长的培养基中摄取维生素B12。因此,当缺乏维生素B12时,藻类使用甲基苯丙胺,但如果维生素B12存在,它可以使用甲基苯丙胺,这是一种更活跃的酶。我们发现维生素B12会导致metE mRNA消失,而且metE mRNA会与固定在小珠子上的维生素B12结合。因此,它很可能受到核开关机制的调节。在这个提议中,我们打算使用许多不同的技术来研究维生素B12(钴胺素)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素很重要,然后对这些核苷酸进行突变,看看这是否会减少代谢物的结合。我们将通过监测mRNA的水平,以及将核糖体开关连接到一种叫做荧光素酶的基因上,来测试这些突变对细胞的影响。荧光素酶的表达可以通过发光来监测。我们还将研究钴胺- mrna复合物的结构。然后我们将确定这些核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、mRNA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,我们将探讨衣藻中另外两个基因也受核糖体开关机制调节的可能性。
项目成果
期刊论文数量(10)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism.
- DOI:10.3389/fpls.2021.708370
- 发表时间:2021
- 期刊:
- 影响因子:5.6
- 作者:Jackson HO;Taunt HN;Mordaka PM;Smith AG;Purton S
- 通讯作者:Purton S
High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform.
- DOI:10.1098/rsif.2015.0216
- 发表时间:2015-05-06
- 期刊:
- 影响因子:0
- 作者:Abalde-Cela S;Gould A;Liu X;Kazamia E;Smith AG;Abell C
- 通讯作者:Abell C
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Alison Smith其他文献
Uncertainty in current and future health wearables
当前和未来健康可穿戴设备的不确定性
- DOI:
- 发表时间:
2018 - 期刊:
- 影响因子:22.7
- 作者:
Bran Knowles;Alison Smith;Forough Poursabzi;D. Lu;Halimat Alabi - 通讯作者:
Halimat Alabi
Perioperative Fluid Management in Surgical Patients: A Review
手术患者围手术期液体管理:综述
- DOI:
- 发表时间:
2022 - 期刊:
- 影响因子:0
- 作者:
Alex Cao;Lillian T Bellfi;J. Schoen;P. Greiffenstein;Alan B Marr;L. Stuke;J. Hunt;R. Pino;Alison Smith - 通讯作者:
Alison Smith
Self-Fulfilling Prophecies, Perceptual Biases, and Accuracy at the Individual and Group Levels
自我实现的预言、感知偏差以及个人和群体层面的准确性
- DOI:
- 发表时间:
1998 - 期刊:
- 影响因子:0
- 作者:
Alison Smith;L. Jussim;J. Eccles;Michelle VanNoy;Stephanie Madon;P. Palumbo - 通讯作者:
P. Palumbo
Uveitis Anterior Asociado a Retinitis Pigmentosa: Reporte de un Caso
前葡萄膜炎与色素性视网膜炎:Reporte de un Caso
- DOI:
10.56172/oftalmica.v22i.39 - 发表时间:
2022 - 期刊:
- 影响因子:0
- 作者:
Alison Smith - 通讯作者:
Alison Smith
Mechanism of decreased forward stroke volume in children and swine with ventricular septal defect and failure to thrive.
患有室间隔缺损和生长障碍的儿童和猪前向输出量减少的机制。
- DOI:
- 发表时间:
1988 - 期刊:
- 影响因子:15.9
- 作者:
W. Corin;M. Swindle;James;F.;Spann;Kiyoharu Nakano;Mary;Frankis;Robert W. W. Biederman;Alison Smith;Ashby;Taylor;Blase A. Carabello - 通讯作者:
Blase A. Carabello
Alison Smith的其他文献
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{{ truncateString('Alison Smith', 18)}}的其他基金
Collaborative Research: Neotoma Paleoecology Database, a Multi-Proxy, International, Community-Curated Data Resource for Global Change Research
合作研究:Neotoma 古生态学数据库,一个用于全球变化研究的多代理、国际、社区策划的数据资源
- 批准号:
1948297 - 财政年份:2020
- 资助金额:
$ 26.12万 - 项目类别:
Continuing Grant
18-BBSRC-NSF/BIO Focusing a quantitative lens on synthetic phototrophic communities
18-BBSRC-NSF/BIO 将定量视角聚焦于合成光养群落
- 批准号:
BB/T010525/1 - 财政年份:2020
- 资助金额:
$ 26.12万 - 项目类别:
Research Grant
(Re)design of the choroplast genome - towards a synthetic organelle
叶绿体基因组的(重新)设计 - 走向合成细胞器
- 批准号:
BB/R01860X/1 - 财政年份:2018
- 资助金额:
$ 26.12万 - 项目类别:
Research Grant
17-ERACoBioTech: MicroalgaE as Renewable Innovative green cell facTories
17-ERACoBioTech:微藻作为可再生创新绿色细胞工厂
- 批准号:
BB/R021694/1 - 财政年份:2018
- 资助金额:
$ 26.12万 - 项目类别:
Research Grant
EKN Tool Assessor: Facilitating the application of innovative tools in the assessment of ecosystem services, green infrastructure and natural capital
EKN Tool Assessor:促进创新工具在生态系统服务、绿色基础设施和自然资本评估中的应用
- 批准号:
NE/P01254X/1 - 财政年份:2016
- 资助金额:
$ 26.12万 - 项目类别:
Fellowship
Collaborative Research: Neotoma Paleoecology Database, Community-led Cyberinfrastructure for Global Change Research
合作研究:Neotoma 古生态学数据库、社区主导的全球变化研究网络基础设施
- 批准号:
1550721 - 财政年份:2016
- 资助金额:
$ 26.12万 - 项目类别:
Continuing Grant
The twilight zone: the initiation of starch degradation in leaves
暮光区:叶子中淀粉降解的开始
- 批准号:
BB/N001389/1 - 财政年份:2016
- 资助金额:
$ 26.12万 - 项目类别:
Research Grant
EarthCubeIA: Collaborative Proposal: Building Interoperable Cyberinfrastructure (CI) at the Interface between Paleogeoinformatics and Bioinformatics
EarthCubeIA:协作提案:在古地理信息学和生物信息学之间的接口处构建可互操作的网络基础设施 (CI)
- 批准号:
1540994 - 财政年份:2015
- 资助金额:
$ 26.12万 - 项目类别:
Standard Grant
Developing platforms for the production of diterpenoids
开发二萜类化合物生产平台
- 批准号:
BB/M018180/1 - 财政年份:2015
- 资助金额:
$ 26.12万 - 项目类别:
Research Grant
14-PSIL Combining Algal and Plant Photosynthesis (CAPP2)
14-PSIL 结合藻类和植物光合作用 (CAPP2)
- 批准号:
BB/M006352/1 - 财政年份:2014
- 资助金额:
$ 26.12万 - 项目类别:
Research Grant
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