Although many proteins require specific interaction with a ligand for their biological activity, little is known about how such proteins bind ligands or how their synthesis is regulated by ligand availability. To understand assembly of ligand-binding proteins, we are studying the pathway and regulation of bacterial cytochrome biosynthesis. As a model, we are studying cytochrome c2 (cyt c2) of the photosynthetic bacterium Rhodobacter sphaeroides. Cyt c2 is a well-characterized, c-type cytochrome that is essential for photosynthesis. In order to function, the cyt c2 precursor protein (preCycA) must be exported to the periplasm, and bind heme. Our long range goal is to define how and where cyt c2 binds its heme ligand, and determine how cyt c2 synthesis is regulated by heme availability. We seek to answer the answer the following questions regarding cyt c2 biosynthesis. 1. How is heme attachment accomplished? We will determine if heme is attached prior or subsequent to removal of the preCycA signal sequence. The heme lyase enzyme (HeLA) will be purified and used to define the cytochrome and cellular factors required for heme attachment. 2. What cytochrome determinants influence cyt c2 synthesis? We will analyze heme attachment to mutant CycA proteins containing substitutions in the heme binding site and to a series of truncated CycA gene products. In addition, CycA substitutions that block export will be analyzed to see if heme attachment and export are coupled. To determine how individual mutations block cyt c2 synthesis, we will compare the conformation of mutant proteins to the wild type CycA gene product. 3. Does HelA attach heme to more than one cytochrome? We will clone the R. sphaeroides HelA structural gene and construct helA mutants. The phenotype of helA mutants will define whether this enzyme attaches heme to more than one c-type cytochrome. 4. What cycA target sites couple cyt c2 synthesis to ligand availability? Our data suggests the heme precursor delta-aminolevulinic acid (ALA) represses cycA transcription. We will define ALA-responsive cycA promoters, and map the cycA transcription will be determined. 5. Does the repressor interact with target sequences in an ALA-dependent fashion? Transcription from ligand-sensitive cycA promoters appears to be repressed by a protein (ChrR) whose activity requires ALA. Trans-acting mutants in the ALA-responsive repressor will be characterized, and the gene defined by these mutations will be identified. To test if ALA is the physiological co-repressor of cycA transcription, the purified protein will be tested for ALA-dependent binding to cycA promoters.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037509-06
Application #
3292789
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-08-01
Project End
1995-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Ranson-Olson, Britton; Jones, Denise F; Donohue, Timothy J et al. (2006) In vitro and in vivo analysis of the role of PrrA in Rhodobacter sphaeroides 2.4.1 hemA gene expression. J Bacteriol 188:3208-18
Tavano, Christine L; Donohue, Timothy J (2006) Development of the bacterial photosynthetic apparatus. Curr Opin Microbiol 9:625-31
Green, Heather A; Donohue, Timothy J (2006) Activity of Rhodobacter sphaeroides RpoHII, a second member of the heat shock sigma factor family. J Bacteriol 188:5712-21
Laguri, Cedric; Stenzel, Rachelle A; Donohue, Timothy J et al. (2006) Activation of the global gene regulator PrrA (RegA) from Rhodobacter sphaeroides. Biochemistry 45:7872-81
Jones, Denise F; Stenzel, Rachelle A; Donohue, Timothy J (2005) Mutational analysis of the C-terminal domain of the Rhodobacter sphaeroides response regulator PrrA. Microbiology 151:4103-10
Anthony, Jennifer R; Warczak, Kristin L; Donohue, Timothy J (2005) A transcriptional response to singlet oxygen, a toxic byproduct of photosynthesis. Proc Natl Acad Sci U S A 102:6502-7
Anthony, Jennifer R; Newman, Jack D; Donohue, Timothy J (2004) Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR. J Mol Biol 341:345-60
Tavano, Christine L; Comolli, James C; Donohue, Timothy J (2004) The role of dor gene products in controlling the P2 promoter of the cytochrome c2 gene, cycA, in Rhodobacter sphaeroides. Microbiology 150:1893-9
Comolli, James C; Donohue, Timothy J (2004) Differences in two Pseudomonas aeruginosa cbb3 cytochrome oxidases. Mol Microbiol 51:1193-203
Anthony, Jennifer R; Green, Heather A; Donohue, Timothy J (2003) Purification of Rhodobacter sphaeroides RNA polymerase and its sigma factors. Methods Enzymol 370:54-65

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