UC Berkeley

Gene expression in eukaryotic cells is an intricate, multi-step process that allows the information encoded in DNA to be translated into proteins, which perform most of the critical functions within the cell. This process is highly regulated at various levels to ensure precise control over which genes are expressed, when, where, and to what extent. The primary stages of gene expression encompass transcription, RNA processing, messenger RNA (mRNA) export, and translation. Among these, transcription within eukaryotic cells is also a complex and tightly regulated process which involves a sophisticated interplay between the transcription machinery, regulatory proteins, chromatin structure, and RNA processing. Gaining a deep understanding of these processes is crucial for unraveling the mechanisms of gene expression regulation and its implications for diseases such as cancer, where normal regulatory mechanisms are frequently disrupted. In this work, we reconstituted nucleosomes and active elongation complex with purified proteins, revealing that eukaryotic RNA polymerase II (RNAP) alone struggles significantly with nucleosome clearance, a finding that contrasts with in vivo observations. To further explore this phenomenon, we employed cryogenic electron tomography (cryo-ET), an advanced imaging technique, to capture intermediate states of RNAP as it transcribes through a nucleosome. Lastly, we examined transcription across nucleosome positioning sequences (NPS) in vivo using two-color confocal microscopy in the fruit fly embryos and assessed nucleosome occupancy employing ATAC-seq (assay for transposase-accessible chromatin with sequencing), revealing that these sequences do not significantly slow down transcription nor position nucleosomes with the same accuracy in vivo.