Bacterial Transcription & Regulation
RNA polymerase (RNAP) is a multi-part protein machine that converts genetic information from DNA to RNA in the complex and highly regulated process of transcription. Transcription has three major stages: initiation, elongation, and termination, each of which is controlled by protein transcriptional factors (TFs) (as shown above). Regulation of transcription is key to bacterial physiology and pathogenesis. Our knowledge about the transcription mechanism mostly relies on studies of the model organisms E. coli and Bacillus subtilis. However, we still have a limited understanding of how lineage-specific characteristics of RNAP from non-model organisms interact with transcriptional factors and how properties of RNAP in these bacteria differ from those in E. coli and B. subtilis. Given that RNAP is a key target for antibiotics, a mechanistic understanding of RNAP in pathogenic bacteria will likely lead to the development of novel antibiotics. Our laboratory is interested in gaining insights into the mechanisms of RNAP action and regulation in Clostridioides difficile, a deadly pathogen in the gut microbiome. To achieve this, we use a variety of approaches, including biochemical assays, Illumina sequencing, cryo-EM, bacterial genetics, and functional genomics. We aim to expand our knowledge and methods to other pathogenic bacteria.
RNA polymerase structure and function
C. difficile RNAP is a potent drug target clinically, but studying this enzyme was a challenge in the field because the pathogen is problematic to grow at scales that yield enough pure enzyme. Our lab uses a synthetic expression system to make recombinant C. difficile RNAP in E. coli. The system provides high yields of C. difficile RNAP suitable for biochemical and structural studies and enables rapid mutagenesis (Cao et al., Nature, 2022). We are exploring the distinctive features of C. difficile RNAP and examining how it is regulated by transcriptional factors and intrinsic DNA and RNA signals throughout the different stages of transcription.
Genome-scale regulation of transcription complexes
We determine the RNAP activity on a genome-wide scale by leveraging the advances of next-generation sequencing. We also develop innovative genome-scale sequencing approaches to construct transcriptional profiling across different combinations of transcriptional factors. Together these methods allow us to generate a comprehensive view of how global transcriptional factors - proteins that regulate transcription across large portions of or entire genes in cells- influence the architecture and function of the transcriptome.
High-throughput screening of transcription inhibitors
We utilize a cutting-edge approach known as fluorescent-aptamer-based assay to closely monitor RNA synthesis in real-time and with high-throughput capabilities. This strategy enables us to efficiently screen compounds that can directly inhibit the activity of RNAP or transcriptional factor alone, or those that disrupt the interactions between the two. Additionally, we will apply this method to investigate the transcription machinery of various pathogenic bacteria for further insight into RNA synthesis.
