Structural study of direct associations between cellular RNA polymerase and regulatory factors during the transcription cycle

Project Summary DNA transcription by cellular and multi-subunit RNA polymerase plays a central role in gene expression. Our investigations have led to significant advancements in understanding the structural basis of DNA transcription that have been achieved through the use of advanced structural biology techniques such as X-ray crystallography and cryo-electron microscopy single particle reconstruction (cryo-EM). Supported by the MIRA grant since 2019, our investigations have expanded and resulted in groundbreaking discoveries concerning the fundamental processes governing gene expression and regulation in bacteria, archaea, and eukaryotes. Notably, our recent cryo-EM studies have shed light on the structural basis of ribosomal RNA transcription by bacterial RNA polymerase, its regulation by DksA/ppGpp, and NusG-dependent transcription pausing. These studies have underscored the importance of analyzing the heterogeneity of transcription complexes, the dynamics of RNA polymerase, and deciphering the multiple states of the transcription complex to understand the long-range impact of transcription factors on RNA polymerase activity regulation. Over the next five years, our research will focus on investigating heterogeneity and dynamics of RNA polymerase transcription using cryo-EM, single-molecule and biochemical approaches, addressing questions that were previously challenging to explore solely through static views of the transcription complex. Specifically, our objectives are to examine the allosteric influence of upstream DNA and bacterial RNA polymerase interactions on DNA opening and transcription initiation, elucidate the mechanisms underlying allosteric and pleiotropic bacterial transcriptional regulation by DksA/ppGpp, analyze bacterial RNA polymerase transcription pausing in an environment closely resembling the intercellular conditions, comprehensively understand the structural basis of the archaeal RNA polymerase transcription cycle, and investigation of eukaryotic RNA polymerase II transcription complexes directly isolated from Drosophila nuclear extract. We anticipate that our proposed project will significantly advance transcription research and enhance our understanding of gene expression across all three domains of life. Moreover, it will contribute to the continued growth of our research program and establish an even greater scientific impact. Gene expression by cellular polymerase is fundamental to all life forms and investigating the transcription process is critical for understanding cell development, maintenance, and disease. The proposed studies regarding the structure and function of cellular RNA polymerase may lead to new avenues for drug discovery and preventing disease.