Effects of Substitutions in a Conserved DX2GR Sequence Motif, Found in Many DNA-dependent Nucleotide Polymerases, on Transcription by T7 RNA Polymerase

The region in bacteriophage T7 RNA polymerase (RNAP) comprising residues 421–425 contains a sequence motif (DX2GR) that is conserved among many DNA-dependent nucleotide polymerases. We have found that alterations in this motif result in enzymes that display weaker retention of the RNA product during transcript initiation, a decreased ability to make the transition to a stable elongation complex, and changes in substrate binding and catalytic activity. Many of these defects are coupled with an altered response to the presence or absence of the non-template strand. The observed constellation of defects supports a role for the motif in interacting with and stabilizing the RNA:DNA hybrid during the early stages of transcript initiation. This is consistent with the position of the motif in a T7 RNAP initiation complex. Although a conserved DX2GR sequence motif is also observed in multisubunit RNAPs, the structural organization of the motif and the manner in which it interacts with the RNA:DNA hybrid in the latter enzymes is different from that in T7 RNAP. However, another element in the multisubunit RNAPs that contains a highly conserved arginine residue may play the same role as R425 in T7 RNAP.