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

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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.