Author

Hao Geng

Date

September 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Environmental and Biomolecular Systems

Institution

Oregon Health & Science University

Abstract

The ResD-ResE two-component signal transduction system is required for the expression of genes involved in anaerobic nitrate respiration in Bacillus subtilis. Response regulator ResD is phosphorylated by the cognate histidine sensor kinase ResE. Phosphorylated ResD (ResD~P) activates transcription of genes including fnr, nasDEF, and hmp. High-resolution hydroxyl radical footprinting assay and mutational analysis of the ResD-controlled promoter regions revealed that two monomers of ResD bind to the TTGTAAN[subscript]3TTN[subscript]4A sequence. ResD binds either to a single site or tandemly to multiple sites as monomer or dimer, and it likely binds in more than one orientation, indicating the flexibility of ResD binding to DNA. E254, V260, Y263, K267, A269 of the C-terminal domain of the ? subunit (?CTD) of RNA polymerase (RNAP) are critical for transcription activation of fnr and nasD. These residues constitute a surface-exposed patch that might interact with ResD during transcription initiation. In contrast, ResD likely interacts with region 4.2 of ?[superscript]A at the hmp promoter. Amino acid residues in the transactivation loop of ResD were shown to be essential for fnr and nasD expression, implying that these residues directly interact with ?CTD; however, DNase I footprinting experiments showed that these residues are likely involved in DNA binding. None of the mutations in the transactivation loop affected hmp expression, leaving ResD regions that interact with RNAP at these promoters unidentified. In addition to the phosphorylation-dependent activation, unphosphorylated ResD upregulates genes of the ResDE regulon in response to oxygen limitation. Furthermore, the NsrR transcriptional regulator participates in activation of of the ResDE regulon, particularly hmp and nasD when nitrate is available. NsrR is likely to contain an iron-sulfur cluster that may sense NO that is generated during nitrate respiration. Interaction of NsrR with NO results in the altered NsrR activity as a transcription regulator.

Identifier

doi:10.6083/M4M61H59

School

OGI School of Science and Engineering

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.