TARGETED MUTAGENESIS IDENTIFIES ASP569 AS A CATALYTICALLY CRITICAL RESIDUE IN T7 RNA-POLYMERASE

In order to look more closely at a well-conserved region in T7 RNA pol ymerase (T7 RNAP) containing, as shown earlier, the functionally essen tial residues Pro-563 and Tyr-571, we used targeted mutagenesis to cha nge those residues within this region that are invariant in all single -subunit RNA polymerases, and characterized the mutant enzymes in vitr o. The most interesting finding of this study was the crucial importan ce of the acidic group of Asp-569. In addition, we have shown that the phenolic ring is the most significant functional group of Tyr-571, wi th the hydroxy group also contributing to promoter binding.