Page 43 - Hormel Report 2021

Figure 1: Cryo-EM structures of class-II cap-dependent transcription activation complex.
to initiate transcription, such as nutritional deprivation conditions. To obtain the structures of holoenzyme and TIC with these factors is crucial for understanding how gene expression is carried out under those envi- ronmental conditions and the underlying differences in promoter recognition and open complex formation by alternative sigma factors.
To elucidate the mechanisms of transcription initiated by activators
2.1. Transcription activation on class-II promoters:
In order to understand the class-II activation mech- anism and investigate whether CAP would induce substantial changes of RNAP during activation, which was not identified by the recent crystal structure of
a class-II transcription activation complex (TAC),
we shall apply cryo-EM to eliminate the impact of crystal packing in X-ray crystallography and obtain the cryo-EM structures of TACs.
2.2. Regulation of initiation by MerR-family activators: To enrich and advance our understanding of tran- scription regulation at initiation, we aim to obtain the structures of TACs with a bound transcription factor, EcmrR protein (one of the MerR-family members),
as well as the promoter DNA that is distinct from the classic ones regulated by CAP, using cryo-EM and X-ray crystallography. The results would help us to understand not only a distinct “twisting” activation mechanism, but also how the MerR-family members respond to xenobiotic effectors and activate the
transcription of multi-drug resistance genes whose products are multidrug efflux pumps, one of three evolved antibi- otic resistance mechanisms in bacteria.
To understand the molecular basis for the regulation of transcription elongation by ATPases
In order to understand how ATPases regulate transcription reactivation,
termination and transcription-associated DNA repair, we shall attempt to obtain structures of the RNAP elongation complexes in association with RapA and other ATPases, such as mfd and UvrD. We shall try to assemble those complexes and determine their cryo-EM structures.
Structural and mechanistic study of transcription and its regulation in Helicobacter pylori
Gastric cancer is a cancer that develops from the inner lining of stomach. The most common cause of gastric cancer is infection by the bacterium Helicobacter pylori (H. pylori), and therefore this bacterium was characterized and identified as a class I carcinogen by the World Health Organization. As the pathogen of gastric cancer, H. pylori has also become a significant target for the prevention of gastric cancer. However, the lack of the structures of the key H. pylori transcription complexes has hindered our understanding of the detailed tran- scription mechanism in H. pylori and precluded the identification of effective drugs to cope with current antibiotic resistance problem. Our long-term goal
is to provide a structural basis for understanding the specific transcription mechanism in H. pylori, characterize RNAP using in vitro transcription assays with or without the relevant antibiotics, and therefore pave the way for discovering effective drugs to eliminate H. pylori infection and reduce the incidence of gastric cancer.
Structural basis of SARS-CoV-2
transcription mechanism
The structures of COVID-19 virus RNA polymerase
in complex with duplex RNA and antiviral drugs
are significant for us to understand the specific replication/transcription mechanism of this novel coronavirus (2019-nCoV or SARS-CoV-2), characterize how antiviral drugs bind to RNA polymerase to inhibit its transcription and therefore inhibit virus replication, and discover novel effective drugs and therapeutic strategies against COVID-19 virus infection.
Figure 2: Cryo-EM structures of sigma28-associated transcription initi- ation complex, EcmrR-associated transcription activation complex, and RapA-associated transcription elongation complex (in clockwise order).
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Publications:
• Shi W., Zhou W., Chen M., Yang Y., Hu Y., Liu B. Structural basis for activation of Swi2/Snf2 ATPase RapA by RNA polymerase. Nucleic Acids Res. 49(18): 10707-10716 (2021).
• Liu C.*, Shi W., Becker S.T., Schatz D.G., Liu B.*,
Yang Y*. Structural basis of mismatch recognition
by a SARS-CoV-2 proofreading enzyme. Science 373(6559):1142-1146 (2021). (* corresponding author)
• Yang Y., Liu C., Zhou W., Shi W., Chen M., Zhang B., Schatz D.G., Hu Y., Liu B. Structural visualization of transcription activated by a multidrug-sensing MerR family regulator. Nat Commun. 12(1): 2702 (2021).