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

Abstract

A series of active elongation complexes of the phage T7 RNA polymerase were obtained through stepwise walking of the polymerase along an immobilized DNA template. Transcripts were radiolabeled at the 16th to 18th residues, and a photocross-linkable 4-thio-UMP was separately incorporated at the 22nd, 24th, 32nd, and 38th residues. Such complexes (up to 51 nucleotides) produced by the incorporation of one nucleotide at a time were isolated and individually subjected to long wave UV cross-linking. Only when the cross-linker 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, regardless 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 contains 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 nascent RNA through the T7 RNA polymerase in elongation complexes.