Structures and functions of Prokaryotic RNA polymerases
Bacterial RNA polymerase
A major theme in my laboratory is understanding how bacterial gene expression is regulated at different stages of transcription by regulatory factors and small molecules. In many cases, transcription initiation, including promoter recognition, double-strand DNA unwinding and promoter escape, is the primary determinant of gene expression. We have revealed structural basis of ribosomal RNA gene expression in E. coli (Shin et al., Nature Commun. 2021) and CTP synthetase gene expression in B. subtilis (Shi et al., Nucleic Acid Res. 2020), by determining the structures of transcription complexes and performing structure-based biochemical assays.
Transcription elongation, pausing and termination as well as RNAP recycling are other critical processes that regulate gene expression, as these processes fine-tune the level of gene expression in response to changes in the environment or cellular state. We determined the cryo-EM structure of the NusG-dependent transcription pausing complex to reveal how the elongation factor NusG temporarily pauses RNA synthesis allosterically (Vishwakarma et al., PNAS 2023). We also investigated how the ATP-dependent motor enzymes RapA in E. coli (Portman et al., Nucleic Acids Res. 2022; Qayyum et al., JBC 2021) and HelD in Mycobacteria smegmatis (Kouba et al., Nature Commun. 2020), enhance gene expression by accelerating RNAP recycling after terminating transcription.
Transcription is a key target for therapeutic interventions and drug development. In collaboration with research groups in academia and pharmaceutical companies, we are developing superior rifampicin derivatives for effective tuberculosis treatment (Rajeswaran et al., ACS Infect Dis. 2022) and a new RNAP inhibitor for the therapeutic treatment of serious human pathogens, such as Staphylococcus aureus (Haupenthal et al., ACS Infect Dis. 2020).
Top) Alternative pathways for open promoter complex formation. Shin et al., Nature Commun. 2021.
Bottom) RNAP conformations associated with transcription elongation, NusG-dependent pausing, and escape from paused transcription. Vishwakarma et al., PNAS 2023.
Archaeal RNA polymerase
The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase II (Pol II) counterpart, comprising a Pol II-like RNA polymerase and the general transcription factors TBP, TFB, TFE and TFS (eukaryotic TBP, TFIIB, TFIIEα and TFS orthologs, respectively). Remarkably, the transcription regulators found in archaeal genomes are closely related to bacterial factors. Therefore, elucidating the transcription mechanism in Archaea, which is a mosaic of bacterial and eukaryotic features, would provide a foundation for unify insights from bacterial, archaeal and eukaryotic systems into basic transcription mechanisms across all three domains of life. We reported the first X-ray crystal structure of the archaeal RNAP from the phyla creachaeota, Sulfolobus solfataricus (Hirata et al., Nature 2008), and a second archaeal RNAP structure from euryarchaeota, another major phyla in Archaea that has retained many features from the last common ancestor of Archaea and Eukaryote (June et al., Nature communications 2014). We also reported the cryo-EM structure of the archaeal RNAP in complex with transcription elongation factor Spt4/5 (Klein et al., Proc Natl Acad Sci U S A 2011). We have investigated how transcription initiation factor (TFEa) and elongation factor (Eta) influence the structure, function and activity of archaeal RNAP by using structural and biochemical approaches (Jun et al., Nature Commun 2020; Marshall et al., PNAS 2022). Structural and mechanistic studies of archaeal RNAP have lagged those of bacteria and eukaryotes; thus, our study is important, as it will reveal the remaining structures and functions of cellular RNAP from all domains of life and its evolution.
A) RNAP structures in the three domains of life.
B) the archaeal RNAP contains Fe-S cluster.
SARS_CoV_2
Since the COVID-19 pandemic started, my group has performed studies on proteins involved in SARS_CoV_2 replication to help end the pandemic by utilizing knowledge and skills gained from our main research. In collaboration with Prof. Joyce, a virologist at Penn State who screened drug candidates inhibiting the proteases of SARS_CoV_2, my group investigated the structure of the main protease (Mpro, also known as 3CLpro) and drug complex by X-ray crystallography (Narayanan et al., Commun Biol. 2022). Extensive structural studies of Mpro have been conducted to determine the mechanism of the proteolytic process by using peptides (8∼20 residues) containing recognition sequences as model substrates. However, there is a lack of structural information regarding the proteolytic process performed by Mpro with polyprotein substrates. We determined the cryo-EM structure of the Mpro and polyprotein complex for the first time to shed light on the interactions of Mpro with different recognition sites on the polyprotein (Narwal et al., J Biol Chem. 2023).
Top) 2D-class averages of the Mpro and polyprotein complex.
Bottom) Cryo-EM density map of the complex (gray transparent) shows the densities of the polyprotein (dashed oval) outside from the recognition site binding Mpro (green and cyan). Narwal et al., JBC 2023.