Biochemical and physical studies of the role of intrinsic metal ions in RNA polymerase from Escherichia coli

Abstract

A variety of nucleotidyl transferases including DNA and RNA polymerases from both prokaryotic and eukaryotic sources are Zn-metalloenzymes. E. coli RNA polymerase (RPase) contains 2 mol of Zn/mol of holoenzyme having a subunit composition of α₂β β's. We have identified that one Zn is located in the β subunit which contains the substrate nucleotide binding site and the other in the β' subunit which possesses the template DNA binding site. While the presence of Zn as an integral part of RPase has been well established, the precise function of the intrinsic metal remains to be elucidated. We have used both in vivo and in vitro metal substitution methods to replace intrinsic Zn with other paramagnetic metals and to probe the functional and structural role of intrinsic metals in RPase.