Mechanisms of transcription and regulation of expression of stable RNAs (precursor ribosomal and 5S) are being studied. Evidence for regulation of stable RNA synthesis by modulation of auxilliary transcriptional protein levels, by RNA polymerase modification and by trans-acting proteins has been obtained in this lab and others. As an initial step in studying the mechanism of regulation of 5S RNA transcription, cloning of the 5S RNA gene, determination of its overall genomic structure and primary sequence is proposed. Development of a cell-free transcription system for 5S RNA to complement the rRNA transcription system already in use in this lab is also proposed. The 5S RNA transcription system will be utilized to examine the molecular mechanism of developmentally regulated shutoff of 5S RNA expression. Emphasis will be on studying the relationship between the mechanisms of the parallel shutdown of 5S and ribosomal RNA resulting from cessation of cellular growth and proliferation. Studies of the structure of the rRNA promoter are ongoing. Deletion mutations constructed and tested for transcriptional activity in vitro have identified a 50 base pair region proximal to the rRNA transcription start site as the promoter. This proposal aims to produce a series of point mutations within this sequence in order to more precisely define the interactions between the promoter and auxilliary transcriptional proteins.

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National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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Colorado State University-Fort Collins
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Fort Collins
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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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