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Analyzing Nitrogenase’s Regulator ‐ NifA’s Interaction with DNA
Author(s) -
Standke Heidi,
Kim Lois,
Medina Michael,
Owens Cedric
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.07560
Subject(s) - nitrogenase , redox , chemistry , cysteine , dna , biochemistry , nitrogen fixation , activator (genetics) , enzyme , gene , nitrogen , organic chemistry
The bacterium Gluconacetobacter diazotrophicus contains an enzyme called nitrogenase which converts dinitrogen gas to ammonia ‐ an essential plant nutrient. The breakdown of dinitrogen has a high energy demand, utilizing 16 ATPs and 16 reducing equivalents. For this reason, nitrogenase is highly regulated. Regulation of nitrogenase is controlled by the protein NifA, a sigma‐54‐activator. NifA is a transcriptional activator that binds to DNA through its DNA binding domain (DBD) to activate nitrogen fixation genes when G. diazotrophicus needs ammonia. NifA also regulates nitrogen fixation depending on whether sufficient redox equivalents are present to support nitrogenase. However, while the role of NifA is known, its mechanism is not fully understood. It is not known if NifA senses oxygen or if it directly senses reducing equivalents. There are two cysteines at the start of NifA’s DNA binding domain. Cysteines are redox active and oxidize to form disulfide bonds which are known to affect protein activity. It is unknown if these cysteines sense oxygen and/or redox conditions and if this potential sensing mechanism affects NifA’s DNA binding. Here, we determine the functional importance of the two cysteines by investigating their redox and O 2 sensing activity, and how the redox state of the cysteines influences NifA’s interaction with DNA. To test the hypothesis that NifA’s DNA binding is regulated by the redox state of the cysteines, two constructs of NifA’s DBD were made: one truncated version containing neither cysteine (No‐Cys) and another containing both cysteines (2‐Cys). Both the No‐Cys and 2‐Cys constructs were cloned into expression plasmids, expressed heterologously in E. coli , purified, and functionally examined through DNA binding assays and biophysical methods. We hypothesized that if NifA is a redox/O 2 sensor, it will only be active when both cysteines are reduced. Our current results indicate that oxidized conditions may be required for proper DNA binding, resulting in K d values in the lower micromolar range. Experiments are currently underway to conclusively determine the redox state of the cysteines and the structure of NifA’s DNA binding domain. Support or Funding Information Chapman University's Undergraduate Student Scholarly Research/Creative Grant Chapman University's Summer Undergraduate Research Fellowship (SURF)