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Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its σ subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of a 32P-labeled phosph-amide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme–oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither the minimal enzyme nor the σ subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the σ subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme. |
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