Revealing the Biophysical Mechanisms Behind Gene Silencing by the Bacterial Immune System, One Transcript at a Time

一次一个转录本揭示细菌免疫系统基因沉默背后的生物物理机制

• 批准号: RGPIN-2019-06520
• 负责人: Milstein, Joshua
• 金额: $ 2.62万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738955
• 关键词: Revealing; Biophysical; Mechanisms; Behind; Gene

We are pioneering biophysical techniques for studying gene expression in the presence of proteins that silence the transcription of newly acquired, foreign DNA. Our focus is both on dissecting the underlying silencing mechanism(s) utilized by these proteins, which remain elusive, and studying how these proteins affect the dynamics of mRNA expression, a possible origin of genetic noise and variability. Our work will contribute to our knowledge of how pathogens evolve and how virulence genes are regulated, with vital implications for biotechnological applications related to expressing foreign genes and/or novel pharmacological substances in bacteria. Bacteria can rapidly adapt to a changing environment by acquiring genes from viruses or other bacteria. Expressing these genes, however, may entail a fitness cost putting the bacteria at a competitive disadvantage or, in the worst case, lead to cell death. The incorporation and eventual expression of foreign DNA is, therefore, carefully controlled. Newly acquired, foreign DNA must, at least initially, be recognized as such and silenced. The protein H-NS, found in common bacteria like E. coli and Salmonella, and Lsr2, found in the pharmaceutical factories Streptomyces, are examples of proteins that target and silence foreign DNA. These proteins are known to somehow interfere with genetic transcription. Bulk biochemical and genomic approaches to understanding gene silencing by H-NS/Lsr2 have been employed for decades. Only recently, with the advent of single-molecule measurements, have researchers begun to gain a fundamental understanding of the biophysical mechanisms through which H-NS/Lsr2, and associated co-regulatory proteins, regulate gene expression. From these experiments, a range of mechanisms have been proposed from cooperatively forming protein-DNA filaments along the genetic sequence, acting as roadblocks, to looping and bridging distant segments of the chromosome, trapping RNAP. While insightful, these single-molecule experiments only probed the interactions between the DNA and the silencing proteins. The DNA sequences were almost always random and no genetic transcription was every actually occurring, leading one to question the biological relevance of such findings. To address these shortcomings, we are developing single-molecule techniques for studying gene silencing on DNA sequences that are actively being expressed. We will be able to study both the relatively rapid dynamics of RNAP procession during a single transcriptional event, as well as the slower dynamics of mRNA production over repeated rounds of transcription-all in the presence of silencing proteins. Simultaneously, we will be able to follow-and apply controlled forces to affect-the procession of RNAP, the production of mRNA, and the conformational state of the substrate DNA. Our program of research will yield an unprecedented level of biophysical detail on the mechanisms and effects of gene silencing.

我们正在开创生物物理技术，用于研究在沉默新获得的外源DNA转录的蛋白质存在下的基因表达。我们的重点是解剖这些蛋白质所利用的潜在沉默机制，这些机制仍然难以捉摸，并研究这些蛋白质如何影响mRNA表达的动态，这是遗传噪音和变异性的可能来源。我们的工作将有助于我们了解病原体如何进化以及毒力基因如何受到调控，并对与表达外源基因和/或细菌中新型药理物质相关的生物技术应用产生重要影响。 细菌可以通过从病毒或其他细菌获得基因来迅速适应不断变化的环境。然而，表达这些基因可能需要适应性成本，使细菌处于竞争劣势，或者在最坏的情况下导致细胞死亡。因此，外来DNA的掺入和最终表达受到仔细控制。新获得的外来DNA必须至少在最初被识别出来并沉默。 蛋白质H-NS，在常见的细菌如大肠杆菌中发现。大肠杆菌和沙门氏菌，以及在制药厂链霉菌中发现的Lsr 2，都是靶向和沉默外源DNA的蛋白质的例子。已知这些蛋白质以某种方式干扰遗传转录。大量的生物化学和基因组的方法来理解基因沉默的H-NS/Lsr 2已经采用了几十年。直到最近，随着单分子测量的出现，研究人员才开始对H-NS/Lsr 2和相关的共调节蛋白调节基因表达的生物物理机制有了基本的了解。从这些实验中，已经提出了一系列的机制，从沿着遗传序列合作形成蛋白质-DNA丝沿着，作为路障，到染色体的环状和桥接远端片段，捕获RNAP。 虽然这些单分子实验很有见地，但它们只探测了DNA和沉默蛋白之间的相互作用。DNA序列几乎总是随机的，没有基因转录是每一个实际发生的，导致人们质疑这些发现的生物相关性。为了解决这些缺点，我们正在开发单分子技术，用于研究正在积极表达的DNA序列上的基因沉默。我们将能够研究在单个转录事件期间RNAP处理的相对快速的动力学，以及在重复的转录轮次中mRNA产生的较慢的动力学-所有这些都是在沉默蛋白的存在下。同时，我们将能够跟踪并施加控制力来影响RNAP的加工、mRNA的产生和底物DNA的构象状态。我们的研究计划将产生一个前所未有的生物物理细节水平的机制和基因沉默的影响。