Structural and functional analysis of ribosome initiation and ribosomal frameshifting.

核糖体起始和核糖体移码的结构和功能分析。

基本信息

批准号：
BB/G008205/1
负责人：
Ian Brierley
金额：
$ 49.36万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2009
资助国家：
英国
起止时间：
2009 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FG008205%2F1
关键词：
Structural functional analysis ribosome initiation

项目摘要

Proteins are polymers of amino acids. The order of the amino acids is governed by the genetic code in the genes of the cell's DNA. In protein synthesis, a gene is first copied into messenger RNA (mRNA) - a single-strand copy of the DNA which retains the code - and then taken to the ribosome. Ribosomes are protein synthesising machines some 25-35nm in diameter and made up of two subunits, one large, one small. The mRNA is loaded linearly between the subunits, whereupon adaptor molecules called transfer RNAs (tRNAs) arrive to decode the mRNA. Each transfer RNA has an amino acid at one end, and at the other, a region which recognises a specific run of three consecutive nucleotides in the mRNA code, a 'triplet'. During protein synthesis, the ribosome moves along the mRNA triplet by triplet and at each step, a tRNA with the appropriate recognition sequence is recruited and the ribosome transfers the tRNA's amino acid to the growing chain of amino acids, the polypeptide. This project will study three aspects of protein synthesis. The first is how mRNA is recruited to the ribosome and fed between the subunits. In mammals, the mRNA interacts with the isolated small subunit of the ribosome in complex with a host of other proteins termed initiation factors. This initiation complex moves along the mRNA, scanning the sequence until the specific triplet that signifies the start of protein synthesis is identified. At this point, the large subunit joins and amino acid polymerisation begins. We wish to characterise in detail the structure of this scanning complex (termed the 48S complex), by purifying initiation complexes paused in the act of scanning along the mRNA, or at the initiation triplet, and study them using microscopy. Our microscope uses electrons rather than light, and is extremely powerful because the short wavelength of electrons allows the visualisation of fine molecular detail. The samples are also frozen at -180C, which keeps them in a natural and stable state, and so the technique is known as 'cryo-electron microscopy' (cryo-EM). The images we obtain will show us the structure of the 48S complex and tell us something of how the various initiation factors allow the small subunit to scan along the mRNA and to start protein synthesis. The second aspect is elongation, when amino acids are added, via tRNAs, to the growing chain. Inside the ribosome are multiple binding sites for tRNAs. A tRNA comes in at one site, binds to its complementary mRNA triplet and transfers its amino acid to the growing chain, moves to an adjacent site to make space for the next tRNA, and finally leaves the ribosome via an exit site. The movement of tRNAs has been hard to analyse at a molecular level. We have identified an mRNA signal termed a pseudoknot which blocks the progress of the ribosome along the mRNA and stalls the ribosome at the point when the tRNAs are moving within the ribosome. We will use cryo-EM to study the structure of ribosomes stalled at a pseudoknot to elucidate the molecular details of tRNA movement. Pseudoknots can also cause the ribosome to change reading frame, that is, to shift from reading one set of triplets (one frame) to reading an overlapping set (another frame), and this leads to a completely new amino acid sequence in the protein. Cryo-EM studies of pseudoknot-stalled ribosomes may thus be informative as to how the ribosome maintains the correct reading frame. The final aspect of the project concerns the structure of ribosomes prepared from mammalian cells. Because the ribosome plays a major role in controlling the rate of protein synthesis, and even which proteins are made, in different cell states the ribosome becomes modified in a variety of ways that affect which proteins are made and how rapidly. By determining structures for ribosomes from cells grown under known conditions we will make the first moves in showing the relationship between ribosome mechanism and regulatory processes in living cells.

蛋白质是氨基酸的聚合物。氨基酸的顺序受细胞DNA基因的遗传密码控制。在蛋白质合成中，首先将基因复制到Messenger RNA（mRNA） - DNA的单链副本，保留该代码，然后将其带入核糖体。核糖体是蛋白质合成的机器，直径约为25-35nm，由两个亚基组成，一个大，一个小。 mRNA在亚基之间线性加载，因此称为转移RNA（TRNA）的衔接子分子到达以解码mRNA。每个转移RNA的一端都有一个氨基酸，另一端具有一个区域，该区域识别mRNA代码中三个连续的核苷酸的特定运行，一个“三重态”。在蛋白质合成过程中，核糖体通过三重态沿mRNA三胞胎移动，在每个步骤中，都会募集具有适当识别序列的tRNA，核糖体将TRNA的氨基酸转移到生长的氨基酸链中，即多肽。该项目将研究蛋白质合成的三个方面。首先是将mRNA募集到核糖体中并在亚基之间喂食。在哺乳动物中，mRNA与复合物中核糖体的分离小亚基与许多其他称为起始因子的宿主相互作用。该起始复合物沿mRNA移动，扫描序列，直到确定蛋白质合成开始的特定三重态。此时，大型亚基连接，氨基酸聚合开始。我们希望详细描述这种扫描复合物（称为48S复合物）的结构，通过净化在沿mRNA扫描的行为中或在启动三重序列中的启动络合物，并使用显微镜对其进行研究。我们的显微镜使用电子而不是光，并且非常强大，因为电子的短波长允许可视化细分子细节。样品还以-180C冷冻，使它们保持自然稳定，因此该技术被称为“冷冻电子显微镜”（Cryo-Em）。我们获得的图像将向我们展示48S复合物的结构，并告诉我们各种起始因子如何允许小亚基沿mRNA扫描并启动蛋白质合成。第二个方面是通过TRNA添加氨基酸到生长的链中时的伸长。核糖体内部是TRNA的多个结合位点。一个位点出现了一个tRNA，与其互补的mRNA三胞胎结合，并将其氨基酸转移到生长的链中，移动到相邻的位点，为下一个trNA腾出空间，最后通过出口部位离开核糖体。 TRNA的运动很难在分子水平上进行分析。我们已经确定了一个称为假诺的mRNA信号，该信号在TRNA在核糖体内移动时，阻断了沿mRNA的核糖体沿mRNA的进展，并使核糖体停滞不前。我们将使用冷冻EM研究停滞在伪诺的核糖体的结构，以阐明tRNA运动的分子细节。伪诺斯还可能导致核糖体改变阅读框，即从读取一组三胞胎（一个框架）转变为读取重叠集（另一帧），这会导致蛋白质中的全新氨基酸序列。因此，关于核糖体如何保持正确的阅读框，对伪恒定的核糖体的冷冻EM研究可能是有益的。该项目的最后一个方面涉及由哺乳动物细胞制备的核糖体的结构。由于核糖体在控制蛋白质合成速率以及制造蛋白质的速率中起着重要作用，因此在不同的细胞状态下，核糖体以各种影响蛋白质产生的蛋白质以及速度的方式进行了修饰。通过确定来自已知条件下生长的细胞的核糖体的结构，我们将采取首次移动，以显示活细胞中核糖体机理与调节过程之间的关系。

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）

Non-canonical translation in RNA viruses.

DOI：
10.1099/vir.0.042499-0
发表时间：
2012-07
期刊：
The Journal of general virology
影响因子：
0
作者：
Firth AE;Brierley I
通讯作者：
Brierley I

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

DOI：
10.1128/jvi.01043-15
发表时间：
2015-08
期刊：
Journal of virology
影响因子：
5.4
作者：
Finch LK;Ling R;Napthine S;Olspert A;Michiels T;Lardinois C;Bell S;Loughran G;Brierley I;Firth AE
通讯作者：
Firth AE

Direct observation of distinct A/P hybrid-state tRNAs in translocating ribosomes.