The objectives of this proposal are to elucidate the structural and functional roles of the intrinsic metal ions in the DNA-dependent RNA polymerases and other regulatory proteins involved in gene transcription. These objectives will be attained by both biochemical and physical approaches, in particular, absorption, fluorescence, NMR, and EPR spectroscopies. RNA polymerases from both prokaryotic and eukaryotic sources have been found to be Zn-metalloenzymes. We propose to study the role of the intrinsic Zn ions in RNA polymerases from E. coli, bacteriophage SP6 and yeast.
Specific aims to be accomplished include: (1) demonstration of the essential role of the intrinsic metal in enzyme catalysis, (2) preparation of apo-enzymes and paramagnetic metal-substituted enzymes, by in vitro and in vivo approaches, (3) comparison of their biochemical and physical properties with those of native enzymes, (4) determination of the proximity relationships between the metal and other active sites on the enzymes, and (5) syncatalytic mapping of the enzymes using the metal as a probe. Recently, we have found that Xenopus transcription factor A(TFIIIA) is a Zn-metalloprotein. The binding of this factor to an intragenic control region in Xenopus oocyte 5S RNA genes is essential for the transcription of these genes by RNA polymerase III. In addition, this factor binds to 5S RNA to form a 7S storage particle, suggesting auto-regulatory mechanism of the 5S RNA gene expression. We propose to investigate the role of Zn ion in factor A with respect to its interaction with DNA and RNA, and its participation in the assembly of a transcriptionally active 5S RNA gene chromatin in vitro. The universal presence of Zn in nucleotidyl transferases and the disturbance in the cell cycle of Zn-deficient cells have been established. Thus, this study not only will elucidate the molecular mechanism by which the intrinsic Zn ions in RNA polymerase and transcriptional factor are involved in gene expression, but also it will shed some light on our understanding of the role of Zn in cell growth, division, and differentiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028057-08
Application #
3275311
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-09-01
Project End
1988-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
8
Fiscal Year
1987
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Tyagi, S C; Wu, F Y (1992) Proximity between nucleotide/dinucleotide and metal ion binding sites in DNA-dependent RNA polymerase from Escherichia coli. Biochemistry 31:6447-53
Wu, F Y; Huang, W J; Sinclair, R B et al. (1992) The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase. J Biol Chem 267:25560-7
Bacolla, A; Wu, F Y (1991) Mung bean nuclease cleavage pattern at a polypurine.polypyrimidine sequence upstream from the mouse metallothionein-I gene. Nucleic Acids Res 19:1639-47
Panth, H; Brenner, M C; Wu, F Y (1991) 1H NMR study of the interaction of ATP with Escherichia coli RNA polymerase containing in vivo-incorporated Co(II). Arch Biochem Biophys 291:307-10
Webster, L C; Zhang, K; Chance, B et al. (1991) Conversion of the E1A Cys4 zinc finger to a nonfunctional His2,Cys2 zinc finger by a single point mutation. Proc Natl Acad Sci U S A 88:9989-93
Shang, Z; Liao, Y D; Wu, F Y et al. (1989) Zinc release from Xenopus transcription factor IIIA induced by chemical modifications. Biochemistry 28:9790-5
Culp, J S; Webster, L C; Friedman, D J et al. (1988) The 289-amino acid E1A protein of adenovirus binds zinc in a region that is important for trans-activation. Proc Natl Acad Sci U S A 85:6450-4
Wu, F Y; Tyagi, S C (1987) Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase. J Biol Chem 262:13147-54
Wu, F Y; Wu, C W (1987) Zinc in DNA replication and transcription. Annu Rev Nutr 7:251-72
Tyagi, S C; Wu, F Y (1987) Synthesis and characterization of fluorescent dinucleotide substrate for the DNA-dependent RNA polymerase from Escherichia coli. J Biol Chem 262:10684-8

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