We are analyzing the mechanism of transcription by eukaryotic RNA polymerase III. We have focused on the polymerase III transcription machinery from the silkworm, Bombyx mori, because there is evidence for tissue-specific regulation of polymerase III activity in this organism. This is a fundamental biological problem. All eukaryotes require the products of correct transcription by polymerase III. Moreover, since it is likely that there are mechanistic features common to all three eukaryotic RNA polymerases, analysis of any one enzyme should also give insight into the others. Using in vitro mutagenesis we have already defined the boundaries of the cisacting elements that direct polymerase III transcription of a silkworm tRNAA1a gene. We have found that the region required for full transcriptional activity of this gene is remarkably large. It includes the coding region, and extends beyond it both upstream and downstream. In the proposed work we will determine whether such large control elements are general features of polymerase III templates, and we will identify the functions they provide. The functional analyses we propose take advantage of our ability to manipulate the components of the Bombyx polymerase III transcription machinery that we have resolved. By varying the elements that act in cis, as well as those that act in trans, we will determine how interactions between these elements lead to correct transcription by polymerase III.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025388-13
Application #
3272978
Study Section
Molecular Biology Study Section (MBY)
Project Start
1978-08-01
Project End
1991-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
13
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Ouyang, C; Martinez, M J; Young, L S et al. (2000) TATA-Binding protein-TATA interaction is a key determinant of differential transcription of silkworm constitutive and silk gland-specific tRNA(Ala) genes. Mol Cell Biol 20:1329-43
Trivedi, A; Young, L S; Ouyang, C et al. (1999) A TATA element is required for tRNA promoter activity and confers TATA-binding protein responsiveness in Drosophila Schneider-2 cells. J Biol Chem 274:11369-75
Young, L S; Ahnert, N; Sprague, K U (1996) Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage. Mol Cell Biol 16:1256-66
Smith, T P; Young, L S; Bender, L B et al. (1995) Silkworm TFIIIA requires additional class III factors for commitment to transcription complex assembly on a 5S RNA gene. Nucleic Acids Res 23:1244-51
Dunstan, H M; Young, L S; Sprague, K U (1994) TFIIIR is an isoleucine tRNA. Mol Cell Biol 14:3588-95
Dunstan, H M; Young, L S; Sprague, K U (1994) tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage. Mol Cell Biol 14:3596-603
Palida, F A; Hale, C; Sprague, K U (1993) Transcription of a silkworm tRNA(cAla) gene is directed by two AT-rich upstream sequence elements. Nucleic Acids Res 21:5875-81
Dieci, G; Duimio, L; Coda-Zabetta, F et al. (1993) A novel RNA polymerase III transcription factor fraction that is not required for template commitment. J Biol Chem 268:11199-207
Young, L S; Dunstan, H M; Witte, P R et al. (1991) A class III transcription factor composed of RNA. Science 252:542-6
Young, L S; Rivier, D H; Sprague, K U (1991) Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors. Mol Cell Biol 11:1382-92

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