Binding of the Priming Nucleotide in the Initiation of Transcription by T7 RNA Polymerase
Unlike DNA polymerases, an RNA polymerase must initiate transcription de novo, that is binding of the initiating (+1) nucleoside triphosphate must be achieved without benefit of the cooperative binding energetics of an associated primer. Since a single Watson-Crick base pair is not stable in solution, RNA polymerases might be expected to provide additional stabilizing interactions to facilitate binding and positioning of the initiating (priming) nucleoside triphosphate at position +1. Consistent with base-specific stabilizing interactions, of the seventeen T7 RNA polymerase promoters in the phage genome, fifteen begin with guanine. In this work, we demonstrate that the purine N7 is important in the utilization of the initial substrate GTP. The fact that on a template encoding AG as the first two bases in the transcript (as in the remaining two of the T7 genome), transcription starts predominantly (but not exclusively) at the G at position +2 additionally implicates the purine O6 as an important recognition element in the major groove. Finally, results suggest that these interactions serve primarily to position the initiating base in the active site. It is proposed that T7 RNA polymerase interacts directly with the Hoogsteen side of the initial priming GTP (most likely via an interaction with an arginine side chain in the protein) to provide the extra stability required at this unique step in transcription.