5S RNA and ribosomal RNA transcription rates, and expression of ribosomal proteins are closely tied to cellular growth rate in order to balance elaboration of ribosomes. This proposal is aimed at studying the mechanism of coordinating expression from these genes, which are transcribed by distinct RNA polymerases. In Acanthamoeba, rRNA transcription is regulated by polymerase I modification, and recent results show that 5S RNA transcriptional shutdown is accompanied by a loss in TFIIlA transcriptional and DNA binding activity in nuclei. The mechanism of this loss is unknown. This study will examine the change in TFIIlA activity by several approaches: The level of TFIIlA protein present in the whole cell will be revealed by Western blot analysis. A change would indicate that there is less TFIIlA in the cell. If so, an investigation of the mechanism for this change will involve cloning the gene for TFIIlA and determining whether TFIIlA mRNA declines in parallel with TFIIlA protein. If so, nuclear run off experiments will determine whether the TFIIlA mRNA level is regulated transcriptionally. If TFIIlA is regulated transcriptionally, an investigation into the mechanism of this regulation will be carried out. First, the promoter of the cloned TFIIlA gene will be dissected using in vitro transcription. If TFIIlA mRNA levels are not regulated transcriptionally, TFIIlA mRNA association with polysomes will be examined to determine if translation is specifically hindered. Alternatively, if the overall cellular level of TFIIlA remains constant, the cellular localization of TFIIlA, eg. as a complex with 5S RNA in the cytoplasm, will be investigated by analyzing the distribution of TFIIlA in nuclei and the cytoplasm, and by determining the amount of 7S RNP and 42S RNP in the cytoplasm at different stages of cellular development and 5S RNA transcriptional activity. If the amount of TFIIIA protein present in nuclei is constant, but only the DNA binding activity of the factor is altered, then TFIIIA must be modified so that it cannot interact with the 5S RNA gene. An investigation of modification will be carried out by structural comparison of TFIIlA from active and inactive cells. Later in Acanthamoeba development, the activity level of TFIIIC is reduced, presumably to shut down other type 2 polymerase III transcription. We will investigate the mechanism of this change using an approach similar to the above study of TFIIIA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026059-18
Application #
2021807
Study Section
Molecular Biology Study Section (MBY)
Project Start
1979-02-01
Project End
1999-12-31
Budget Start
1997-01-01
Budget End
1998-12-31
Support Year
18
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
112617480
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Polakowski, Nicholas; Paule, Marvin R (2002) Purification and characterization of transcription factor IIIA from Acanthamoeba castellanii. Nucleic Acids Res 30:1977-84
Matthews, J L; Zwick, M G; Paule, M R (1995) Coordinate regulation of ribosomal component synthesis in Acanthamoeba castellanii: 5S RNA transcription is down regulated during encystment by alteration of TFIIIA activity. Mol Cell Biol 15:3327-35
Radebaugh, C A; Matthews, J L; Geiss, G K et al. (1994) TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription fac Mol Cell Biol 14:597-605
Imboden, M A; Matthews, J L; Lofquist, A K et al. (1992) An exonuclease requiring an intact helical stem for specificity produces the 3' end of Acanthamoeba castellanii 5 S RNA. J Biol Chem 267:24601-10
Perna, P J; Harris, G H; Iida, C T et al. (1992) The start site of the Acanthamoeba castellanii ribosomal RNA transcription unit. Gene Expr 2:71-8
Zwick, M G; Imboden, M A; Paule, M R (1991) Specific transcription of an Acanthamoeba castellanii 5S RNA gene in homologous nuclear extracts. Nucleic Acids Res 19:1681-6
Zwick, M G; Wiggs, M; Paule, M R (1991) Sequence and organization of 5S RNA genes from the eukaryotic protist Acanthamoeba castellanii. Gene 101:153-7
Paule, M R; Bateman, E; Hoffman, L et al. (1991) Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii. Mol Cell Biochem 104:119-26
Kownin, P; Bateman, E; Paule, M R (1988) Effects of single-base substitutions within the Acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I. Mol Cell Biol 8:747-53
Kownin, P; Bateman, E; Paule, M R (1987) Eukaryotic RNA polymerase I promoter binding is directed by protein contacts with transcription initiation factor and is DNA sequence-independent. Cell 50:693-9

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