The aim of the proposed work is to investigate the mechanism of transcription by silkworm RNA polymerase III in vitro. Much of our effort will focus on a novel transcription component, Factor X, that our laboratory has recently isolated. This factor is an essential part of the machinery that transcribes eukaryotic tRNA and 5S genes. It is generally assumed that such machinery is composed entirely of polypeptides. The remarkable feature of Factor X is that it appears to be an RNA rather than a protein. The finding of an RNA with transcription activity is unprecedented. The discovery of Factor X came from our systematic fractionation of the components required for tRNA and 5S RNA transcription in vitro. It is distinct from the components previously resolved by our laboratory and others: polymerase III, and the transcription factors A, B, C, and D. Factor X appears to be a nucleic acid because it is resistant to heat, detergent, phenol, and protease, but sensitive to nuclease. It appears to be an RNA because it is resistant to DNAse, but sensitive to RNAse and alkali.
The specific aims of this proposal are: (1) to identify the critical segments of the large tRNA(Ala)C promoter that are essential for function, (2) to purify Factor X, as well as the other silkworm transcription factors in order to establish their identities unambiguously, (3) to assign specific functions to Factor X and the other transcription factors by (a) determining whether they participate in factor-template and factor-factor interactions, (b) determining which phase of the cell cycle requires Factor X, and (c) constructing mutant versions of Factor X to test specific hypotheses for its function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025388-15
Application #
3272979
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1978-08-01
Project End
1994-07-31
Budget Start
1992-08-01
Budget End
1993-07-31
Support Year
15
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Schools of Arts and Sciences
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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