The goal of the proposed research is to obtain a complete understanding at the molecular level of a complex regulatory circuit which controls nitrogen metabolism in Neurospora crassa. The multiple functions of the global-acting regulatory protein NIT2, which shares homology with human transcription factors GATA-1, -2, -3, and -4 will be examined in detail. The function and regulation of trans- acting nitrogen regulatory proteins, NIT2, NIT4, and NMR will be elucidated by site-directed and conventional mutagenesis, followed by analyses both in vitro and in vivo. A major focus is upon multiple functional protein-protein interactions in which the global-acting NIT2 participates, between the NIT2 and NMR regulatory proteins and between the NIT2 and NIT4 proteins. These interactions will be investigated by direct biochemical studies , by mutational analysis, and by use of the yeast two-hybrid system. The exact requirements for DNA binding by the pathway-specific NIT4 protein will be determined, and the nature of the NIT4 dimerization domain will be examined. NIT2 and/or NIT4 binding elements will be inserted into the promoter of a new test gene to assess their individual and joint function. Factors responsible for mediating glutamine-dependent nitrogen catabolite repression will be identified by genetic and biochemical studies, and a potential post-translational modification of NIT2 or NMR will be examined. A combined molecular and genetic analysis will be used to understand the molecular basis for the autogenous control of gene expression exerted by the enzyme nitrate reductase, which may involve interaction in a complex feedback loop with the transcription factor NIT4. The function of two new GATA factors will be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023367-22
Application #
6018471
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Anderson, James J
Project Start
1977-01-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
22
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Ohio State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
Gao-Rubinelli, Fei; Marzluf, George A (2004) Identification and characterization of a nitrate transporter gene in Neurospora crassa. Biochem Genet 42:21-34
Mo, Xiaokui; Marzluf, George A (2003) Cooperative action of the NIT2 and NIT4 transcription factors upon gene expression in Neurospora crassa. Curr Genet 42:260-7
Harrison, Kelly A; Marzluf, George A (2002) Characterization of DNA binding and the cysteine rich region of SRE, a GATA factor in Neurospora crassa involved in siderophore synthesis. Biochemistry 41:15288-95
Feng, B; Haas, H; Marzluf, G A (2000) ASD4, a new GATA factor of Neurospora crassa, displays sequence-specific DNA binding and functions in ascus and ascospore development. Biochemistry 39:11065-73
Zhou, L; Marzluf, G A (1999) Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa. Biochemistry 38:4335-41
Tao, Y; Marzluf, G A (1999) The NIT2 nitrogen regulatory protein of Neurospora: expression and stability of nit-2 mRNA and protein. Curr Genet 36:153-8
Feng, B; Marzluf, G A (1998) Interaction between major nitrogen regulatory protein NIT2 and pathway-specific regulatory factor NIT4 is required for their synergistic activation of gene expression in Neurospora crassa. Mol Cell Biol 18:3983-90
Zhou, L W; Haas, H; Marzluf, G A (1998) Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa. Mol Gen Genet 259:532-40
Tao, Y; Marzluf, G A (1998) Synthesis and differential turnover of the CYS3 regulatory protein of Neurospora crassa are subject to sulfur control. J Bacteriol 180:478-82
Coulter, K R; Marzluf, G A (1998) Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa. Curr Genet 33:395-405

Showing the most recent 10 out of 69 publications