Structures and functions of Prokaryotic RNA polymerases

Bacterial RNA polymerase

Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. We reported that E. coli RNAP prepared from a co-overexpression system can be used for crystallization and structure determination (Murakami, J Biol Chem 2013). We determined the crystal structure of E. coli RNAP containing clinically important rifamycin (RIF) resistant mutations in apo-form and also in complex with rifampin (RMP). Our study revealed the mechanism of RIF resistance by RNAP mutation in molecular level (Molodtsov et al., Mol Microbiol 2017).

Bacterial RNAP is one of the best targets for developing new antibiotics. In collaboration with research groups in academia and the pharmaceutical companies, we are developing superior rifampicin-derivatives for effective tuberculosis treatment (Molodtsov et al., J Med Chem 2013) and developing a new RNA polymerase inhibitor as a broad-spectrum antibiotic for therapeutic treatment of serious human pathogens (Molodtsov et al., J Med Chem 2015).

In addition to E. coli RNAP, we determined several structures of Thermus thermophilus transcription initiation complexes including the de novo and the initially transcribing complex containing 6-mer RNA, providing structural basis of abortive transcription, transcription pausing and also releasing the σ factor from the RNAP core enzyme (Basu et al., J Biol Chem 2014). We also determined the crystal structure of RNAP engaged in reiterative transcription from the pyrG promoter and revealed that the reiterative transcript detours from the dedicated RNA exit channel and extends toward the main channel of the enzyme (Murakami, Shin et al., PNAS 2017).

A) X-ray crystal structure of E. coli RNAP σ70 holoenzyme.

B) Proposed mechanism for RIF resistance by the S531L mutation in which RMP binding induces disordering of fork loop 2.

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).

A) RNAP structures in the three domains of life.

B) the archaeal RNAP contains Fe-S cluster.

Bacteriophage N4 RNA polymerase

We has been investigating the transcription mechanism of the bacteriophage N4 RNAP, which belongs to the T7-like family and also to the A-family of DNA polymerase. Using X-ray crystallographic structures, we revealed the mechanisms of hairpin-form promoter DNA recognition (Gleghorn et al., Mol Cell 2008) and also nucleotide substrate recognition by the N4 phage RNAP (Gloghorn et al., Proc Natl Acad Sci 2011). We also developed a unique method called “time-dependent soak-trigger-freeze X-ray crystallography” to monitor the nucleotidyl transfer reaction in real time at atomic resolution (Basu and Murakami, J Biol Chem 2013).

A) Structure of the N4 virion-RNAP and promoter DNA complex.

B) Experimental scheme of the time-dependent soak-trigger-freeze X-ray crystallography.