Two site contact of elongating transcripts to phage T7 RNA polymerase at C-terminal regions

A series of active elongation complexes of the phage T7 RNA polymerase were obtained through stepwise walking of the polymerase along an immobilized D NA template. Transcripts were radiolabeled at the 16th to 18th residues, an d a photocross-linkable 4-thio-UMP was separately incorporated at the 22nd, 24th, 32nd, and 38th residues. Such complexes (up to 51 nucleotides) produ ced by the incorporation of one nucleotide at a time were isolated and indi vidually subjected to long wave UV cross-linking. Only when the cross-linke r was positioned at the 3'-end (-1) of the elongating RNA and 8 nucleotides upstream (-9), was the RNA substantially cross-linked to the polymerase, r egardless of how far it was from the 5'-end of the transcripts. Linkage of the 3'-end residue was mapped to the Thr(636)-Met(666) region, which contai ns nucleotide-binding sites. The -9 residue was cross-linked to the Ala(724 )-Met(750) region rather than to the N-terminal region. These two contacts were maintained throughout the elongation complexes and reveal a route of n ascent RNA through the T7 RNA polymerase in elongation complexes.