Defining how cells regulate the uptake and efflux of transition metals such as Zn and Cu is a key component in elucidating cellular mechanisms of metal homeostasis. Bacterial model systems provide paradigms for understanding metal-responsive gene regulation. In E. coli, the metalloregulator ZntR senses Zn excess and activates Zn efflux, while Zur senses Zn sufficiency and represses Zn uptake, to keep this essential metal at appropriate physiological levels in the cell. CueR, a homolog of ZntR, senses intracellular Cu to activate Cu efflux/detoxification genes to keep this toxic metal minimal. The long-term goal here is to understand how metal regulation in the cell can be manipulated for preventive and therapeutic purposes. Toward this goal, the PI has established an internationally unique research program that applies and develops advanced single- molecule single-cell approaches to interrogate and understand the mechanisms of bacterial metal regulation both in vitro and in live cells, which are further enhanced by bulk biochemical/biophysical and protein/genetic engineering approaches and established collaborations with biologists. The research has led to discoveries of first-of-their-kind mechanisms of Cu/Zn-responsive transcriptional regulation, but new questions also emerged. The objective of this renewal is to continue this program, as well as elucidate the mechanism that couples CueR/ZntR regulation to DNA mechanical tension and the mechanism of Zur?s biphasic unbinding kinetics from DNA, two novel phenomena the PI recently discovered. The premise of this research comprises the importance of (bacterial) metal regulation in biology, the discovered novel and broadly relevant regulation mechanisms, and the power of combining single-molecule/cell and bulk measurements. The proposed research contains two specific aims, each with sub-aims: 1) Identify the mechanism of DNA-mechanical-tension?coupled transcription regulation by CueR/ZntR.
This aim will test hypotheses based on the discoveries that CueR/ZntR?s unbinding from DNA is modulated by chromosome condensation in cells and that CueR/ZntR can control RNAP actions on DNA. 2) Identify the mechanism of biphasic unbinding kinetics of Zur from DNA.
This aim will test hypotheses regarding the preliminary results that apo/holo-Zur shows biphasic (i.e., repressed followed by facilitated) unbinding kinetics from DNA with increasing intracellular protein concentrations. The research is significant because it will elucidate novel molecular mechanisms of metalloregulators in regulating metal efflux and uptake, as well as provide fundamental knowledge about cell biology of metals in general, for identifying causes or developing preventions of diseases that involve similar regulation processes, and for helping the development of (bio)chemical strategies to manipulate bacterial Zn/Cu regulation to impair pathogen growth. The research is innovative because it applies/develops novel single-molecule manipulation, imaging, and analysis methods, and introduce new mechanistic concepts in transcription regulation.

Public Health Relevance

The proposed research is relevant to public health because the mechanisms identified will lead to the development of (bio)chemical strategies to manipulate Zn/Cu regulation in Gram-negative bacteria, which are related to many human pathogens. These strategies can be used to effect limitation of the essential micronutrient Zn, increase the toxicity of Cu, and thus restrict the growth of invading pathogens. Thus, the proposed research is relevant to the part of NIGMS?s mission that pertains to laying the foundation for disease treatment and prevention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM109993-06
Application #
9973160
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2014-07-01
Project End
2023-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
6
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Chen, Tai-Yen; Cheng, Yu-Shan; Huang, Pei-San et al. (2018) Facilitated Unbinding via Multivalency-Enabled Ternary Complexes: New Paradigm for Protein-DNA Interactions. Acc Chem Res 51:860-868
Chandrangsu, Pete; Loi, Vu Van; Antelmann, Haike et al. (2018) The Role of Bacillithiol in Gram-Positive Firmicutes. Antioxid Redox Signal 28:445-462
Santiago, Ace George; Chen, Tai-Yen; Genova, Lauren A et al. (2017) Adaptor protein mediates dynamic pump assembly for bacterial metal efflux. Proc Natl Acad Sci U S A 114:6694-6699
Shin, Jung-Ho; Helmann, John D (2016) Molecular logic of the Zur-regulated zinc deprivation response in Bacillus subtilis. Nat Commun 7:12612
Yang, Feng; Chen, Tai-Yen; Krzemi?ski, ?ukasz et al. (2016) Single-molecule dynamics of the molecular chaperone trigger factor in living cells. Mol Microbiol 102:992-1003
Chen, Tai-Yen; Jung, Won; Santiago, Ace George et al. (2015) Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution. J Phys Chem B 119:14451-9
Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed et al. (2015) Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription. Proc Natl Acad Sci U S A 112:13467-72
Chen, Tai-Yen; Santiago, Ace George; Jung, Won et al. (2015) Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells. Nat Commun 6:7445