Page 29 - Annual Report 2020

1. To elucidate the mechanisms of transcription initiation with alternative sigma factors
In bacteria, there are seven well identified sigma factors. σ70 is the primary sigma factor, which initi- ates the expression of most genes, while other six ones are the alternative sigma factors in response to different environmental conditions. Previous structural studies of the holoenzyme and tran- scription initiation complex (TIC) with σ70, σ54, σ38, and σ24 have shed light on RNAP-mediated gene expressions under normal conditions, under ni- trogen-limited conditions, under starvation stress, and under heat stress, respectively. However, the structures of those complexes with σ32, σ28, and σ19 are still unknown. These factors are recruited un- der different stress conditions to initiate transcrip- tion, 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 differ- ences in promoter recognition and open complex formation by alternative sigma factors.
2. To elucidate the mechanisms of transcription initiated by activators
2.1. Transcription activation on class-II promot-
ers: In order to understand the class-II activation
mechanism 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 acti-
vation complex (TAC), we shall apply cryo-EM to
eliminate the impact of crystal packing in X-ray
crystallography and obtain the cryo-EM structures bacterium was characterized and identified as a
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 transcription mechanism in H. pylori and precluded the identification of effective drugs to cope with current antibiotic resistance problems. Our long-term goal is to provide a structural basis for understanding the specific transcription mech- anism in H. pylori, characterize RNAP using in vitro transcription assays with or without the relevant antibiotics, and therefore pave the way for discov- ering effective drugs to eliminate H. pylori infection and reduce the incidence of gastric cancer.
5. 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), charac- terize 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.
2.2. Regulation of initiation by MerR-family activa- tors: To enrich and advance our understanding of transcription regulation at initiation, we aim to ob- tain the structures of TACs with a bound transcrip- tion 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 multidrug resistance genes whose products are multidrug efflux pumps, one of three evolved antibiotic resistance mechanisms in bacteria.
3. To understand the molecular basis for the regulation of transcription elongation by ATPases
In order to understand how ATPases regulate transcription reactivation, termination and tran- scription-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.
4. 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
of TACs.
class I carcinogen by the World Health Organiza- tion. As the pathogen of gastric cancer,
THE HORMEL INSTITUTE // UNIVERSITY OF MINNESOTA PG 29