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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=716185
• 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 transcriptionall in the presence of silencing proteins. Simultaneously, we will be able to followand apply controlled forces to affectthe 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.

我们正在开拓生物物理技术来研究基因表达的蛋白质，沉默新获得的转录，外源DNA的存在。我们的重点是剖析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，这是遗传噪声和变异的可能来源。我们的工作将有助于我们了解病原体是如何进化的以及毒力基因是如何被调控的，这对在细菌中表达外源基因和/或新药理物质的生物技术应用具有重要意义。