Structural and functional analysis of ribosome initiation and ribosomal frameshifting.

蛋白质是氨基酸的聚合物。氨基酸的顺序是由细胞DNA基因中的遗传密码决定的。在蛋白质合成中，一个基因首先被复制成信使RNA （mRNA）——一种保留密码的DNA单链拷贝——然后被带到核糖体。核糖体是蛋白质合成机器，直径约25-35纳米，由两个亚基组成，一个大，一个小。mRNA在亚基之间线性加载，随后称为转移rna （trna）的衔接分子到达并解码mRNA。每个转移RNA的一端都有一个氨基酸，另一端则有一个区域，该区域可以识别mRNA编码中三个连续核苷酸的特定序列，即“三联体”。在蛋白质合成过程中，核糖体沿着mRNA的三联体移动，在每一步中，一个具有适当识别序列的tRNA被招募，核糖体将tRNA的氨基酸转移到氨基酸的生长链上，即多肽。本项目将研究蛋白质合成的三个方面。首先是mRNA如何被招募到核糖体并在亚基之间传递。在哺乳动物中，mRNA与核糖体分离的小亚基相互作用，并与一系列称为起始因子的其他蛋白质相互作用。这个起始复合物沿着mRNA移动，扫描序列，直到识别出标志着蛋白质合成开始的特定三联体。在这一点上，大亚基连接和氨基酸聚合开始。我们希望详细描述这种扫描复合体（称为48S复合体）的结构，通过纯化沿mRNA或起始三重体扫描时暂停的起始复合体，并使用显微镜研究它们。我们的显微镜使用的是电子而不是光，它的功能非常强大，因为电子的波长很短，可以看到精细的分子细节。这些样品也被冷冻在零下180摄氏度，这使它们保持在自然和稳定的状态，因此这项技术被称为“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着mRNA扫描并开始蛋白质合成。第二个方面是延伸，当氨基酸通过trna添加到生长链上时。核糖体内部有多个trna结合位点。一个tRNA从一个位点进入，与互补的mRNA三联体结合，并将其氨基酸转移到生长链上，移动到邻近的位点为下一个tRNA腾出空间，最后通过出口位点离开核糖体。trna的运动很难在分子水平上进行分析。我们已经确定了一种被称为假结的mRNA信号，它阻断了核糖体沿着mRNA的进展，并在trna在核糖体内移动时阻止核糖体。我们将使用冷冻电镜研究在假结处停滞的核糖体的结构，以阐明tRNA运动的分子细节。假结也可以导致核糖体改变阅读框，即从阅读一组三联体（一帧）转变为阅读重叠组（另一帧），这导致蛋白质中出现全新的氨基酸序列。因此，对假结停滞核糖体的冷冻电镜研究可能有助于了解核糖体如何维持正确的阅读框。该项目的最后一个方面涉及从哺乳动物细胞制备的核糖体的结构。因为核糖体在控制蛋白质合成的速度，甚至是蛋白质的合成方面起着重要的作用，所以在不同的细胞状态下，核糖体会以各种方式进行修饰，从而影响蛋白质的合成和速度。通过确定在已知条件下生长的细胞中核糖体的结构，我们将在展示活细胞中核糖体机制和调节过程之间的关系方面迈出第一步。

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.