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.
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