Characterizing fundamental principles of translational control in the bacterial model organism E. coli using a novel deep-sequencing technique

基因表达是活细胞中最基本的过程，分两步进行：通过RNA聚合酶（RNAP）将基因的DNA序列转录成mRNA，以及通过核糖体将mRNA模板翻译成蛋白质。在细菌中，这两个步骤是耦合的，因为核糖体在转录过程中启动mRNA分子的翻译（转录-翻译偶联）测序技术的进步（深度测序）通过提供mRNA上单个翻译核糖体的位置的全基因组视图，并转录RNAP，改变了我们对翻译和转录控制的理解。这使我们能够确定合成了哪些蛋白质以及每种蛋白质的数量，以及核糖体和RNAP在哪里暂停。然而，这些方法不能测量单个mRNA分子上的机器之间的距离。因此，我们不知道翻译mRNA分子的多个核糖体之间的间隔，或者前导核糖体和转录RNAP之间的间隔。因此，我们缺乏基本的参数来描述和理解翻译和转录-翻译耦合。我正在开发一种新的深度测序方法（ASITE），使我们能够测量核糖体的确切位置及其相对于彼此的间距，以及在E.杆菌使用ASITE，我们会问：翻译起始是随机的，还是发生在一起启动的核糖体包的爆发？mRNA在翻译过程中调节核糖体间距吗？如何调节？细胞是否协调核糖体和RNAP的距离，如果是的话，是什么机制？这些研究可能会对在单个mRNA水平上理解细菌的翻译产生变革性的影响。此外，这里开发的ASITE方法将广泛适用于真核生物的翻译研究，包括人类细胞和病毒翻译程序。

Gene expression, the most fundamental process in living cells, is carried out in two steps: transcription of the DNA sequence of a gene into mRNA by RNA polymerase (RNAP), and translation of the mRNA template into protein by ribosomes. In bacteria, both steps are coupled as ribosomes initiate translation of mRNA molecules during their transcription (transcription-translation coupling).Advances in sequencing technology (deep sequencing) transformed our understanding of translational and transcriptional control by providing a genome-wide view of the positions of individual translating ribosomes on mRNAs, and transcribing RNAPs. This enabled us to determine what proteins are synthesized and how much of each, as well as where the ribosome and RNAP are pausing. However, these methods cannot measure distances between machineries on single mRNA molecules. Thus, we do not know the spacing between multiple ribosomes translating an mRNA molecule, or between the lead ribosome and transcribing RNAP. Therefore, we lack fundamental parameters to describe and understand translation and transcription-translation coupling.I am developing a novel deep-sequencing method (ASITE) that allows us to measure both the exact positions of ribosomes and their spacing relative to each other and to RNAP on individual mRNAs in E. coli. Using ASITE, we will ask: Is translation initiation stochastic, or does it occur in bursts with packs of ribosomes initiating together? Do mRNAs regulate ribosome spacing during translation, and how? Do cells coordinate ribosome and RNAP distances, and if yes, what are the mechanisms to do so? These studies will likely have a transformative impact on understanding translation in bacteria at the single mRNA level.Furthermore, the ASITE method developed here will be widely applicable to translation research in eukaryotic organisms, including human cells and viral translation programs.