This is a proposal to study the mechanism of rRNA transcription and its regulation in eukaryotes. An in vitro transcription system made up of cloned rDNA, homogeneous RNA polymerase I (RNAP I) and an auxilliary protein (transcription initiation factor(s)-TIF) is being utilized: (1) to determine the number of TIFs, and to purify them to homogeneity, (2) to determine in greater detail the role played by the TIF in initiation of transcription, (3) to determine how the TIF and RNAP I interact with each other and with the template and (4) to describe kinetically the events involved in the assembly of the initiation complex. DNA footprinting and chemical modification-protection experiments will be used to determine protein-DNA contacts. Chemical cross-linking experiments will be used to determine protein-protein interactions. Kinetic measurements will be used to determine the kinetic mechanism of initiation. Monoclonal antibodies to the RNAP I subunits and the TIF(s) will be produced to aid in the analysis. Transcription of rRNA gene expression is developmentally regulated in Acanthamoeba by modification of RNAP I. Experiments to identify the chemical nature of the modification and the enzyme catalyzing it are proposed. Exactly which step in the process of initiation is affected by the modification is also to be studied utilizing the techniques proposed to study the mechanism of initiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022580-12
Application #
3271211
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1979-04-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
12
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Type
Schools of Arts and Sciences
DUNS #
112617480
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
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Bric, Anka; Radebaugh, Catherine A; Paule, Marvin R (2004) Photocross-linking of the RNA polymerase I preinitiation and immediate postinitiation complexes: implications for promoter recruitment. J Biol Chem 279:31259-67
Georgel, Philippe T; Robert, Charles H (2002) Differential core histone binding behavior: RNA polymerase I promoter region vs 5S rDNA positioning DNA sequences. Cell Biochem Biophys 37:1-13
Al-Khouri, Anna Maria; Paule, Marvin R (2002) A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding. Mol Cell Biol 22:750-61
Polakowski, Nicholas; Paule, Marvin R (2002) Purification and characterization of transcription factor IIIA from Acanthamoeba castellanii. Nucleic Acids Res 30:1977-84
Slodzinski, M K; Blaustein, M P (1998) Physiological effects of Na+/Ca2+ exchanger knockdown by antisense oligodeoxynucleotides in arterial myocytes. Am J Physiol 275:C251-9
Radebaugh, C A; Kubaska, W M; Hoffman, L H et al. (1998) A novel transcription initiation factor (TIF), TIF-IE, is required for homogeneous Acanthamoeba castellanii TIF-IB (SL1) to form a committed complex. J Biol Chem 273:27708-15
Geiss, G K; Radebaugh, C A; Paule, M R (1997) The fundamental ribosomal RNA transcription initiation factor-IB (TIF-IB, SL1, factor D) binds to the rRNA core promoter primarily by minor groove contacts. J Biol Chem 272:29243-54
Gong, X; Radebaugh, C A; Geiss, G K et al. (1995) Site-directed photo-cross-linking of rRNA transcription initiation complexes. Mol Cell Biol 15:4956-63

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