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

我们正在开创生物物理技术，在蛋白质存在的情况下研究基因的表达，这些蛋白质可以沉默新获得的外来DNA的转录。我们的重点是剖析这些蛋白质利用的潜在沉默机制(S)，以及研究这些蛋白质如何影响基因表达的动态，这可能是遗传噪声和变异性的来源。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受到调控的，这对在细菌中表达外源基因和/或新的药理物质的生物技术应用具有重要意义。细菌可以通过从病毒或其他细菌中获取基因来快速适应不断变化的环境。然而，表达这些基因可能需要付出适合度成本，使细菌处于竞争劣势，或者在最糟糕的情况下，导致细胞死亡。因此，外源DNA的加入和最终的表达受到谨慎的控制。新获得的外来DNA必须至少在一开始就被承认是这样的，并保持沉默。*在大肠杆菌和沙门氏菌等常见细菌中发现的H-NS蛋白，以及在制药厂链霉菌中发现的Lsr2，都是靶向并沉默外源DNA的蛋白质。众所周知，这些蛋白质会以某种方式干扰基因转录。用生物化学和基因组学方法研究H-NS/Lsr2的基因沉默已有几十年的历史。直到最近，随着单分子测量的出现，研究人员才开始对H-NS/Lsr2及其相关共调控蛋白调控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，从沿着基因序列协同形成蛋白质-DNA细丝，充当障碍，到环路和桥接染色体的远处片段，捕获RNAP。虽然这些单分子实验很有洞察力，但它们只探索了DNA和沉默蛋白之间的相互作用。DNA序列几乎总是随机的，没有任何基因转录是实际发生的，这导致人们质疑这些发现的生物学相关性。为了解决这些缺点，我们正在开发单分子技术来研究活跃表达的DNA序列上的基因沉默。我们将能够研究在单个转录事件中RNAP过程的相对快速动力学，以及在沉默蛋白存在的情况下，在重复几轮转录中mRNA产生的较慢动力学。同时，我们将能够跟踪和施加受控力量来影响RNAP的过程、mRNA的产生和底物DNA的构象状态。我们的研究计划将在基因沉默的机制和影响方面产生前所未有的生物物理细节。

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.