Elongating transcript binding to T7 RNA polymerase

Abstract

In order to study transcription elongation mechamism of phage T7 RNA polymerase, stepwise walking of RNA polymerase was constructed by immobilizing biotinylated DNA template with streptavidin bead. Series of active and stable elongation complexes were obtained. Transcripts were radiolabeled at the 16th to 18th residues, and a photocross-linkable 4-thio-UMP was incorporated at the 22nd, 24th, 32nd, and 38th residues separately to identify the protein-RNA interaction in elongation complex of T7 RNA polymerase. Such complexes (up to 51 nt1) produced by the incorporation of a nucleotide at a time were isolated and subjected to long-wave UV cross-linking individually. As a result, only when the cross-linker is positioned at the 3` end (-1) of the elongating RNA and 8 nucleotides upstream (-9), it is substantially cross-linked to the polymerase, regardless how far it is from the 5 end of transcripts. Above two RNA binding sites of T7 RNA polymerase was determined using trypsin, hydroxylamine, 2-nitro-4-thiocyano benzoic acid and cyanogen bromide. Linkage of the 3`-end residue is mapped to the 636Thr-666Met region, which contains nucleotide-binding sites as previously identified. The 9 residue is cross-linked to the C-terminal 724Ala-750Met region rather than an N-terminal region as other results and models. These two contacts are maintained throughout the elongation complexes, and reveal a route of a growing RNA chain on the polymerase structure. The results suggest that active site of T7 RNA polymerase is conserved during transcription processes from initiation to elongation. Conformation change of elongation complex from initiation to elongation is also inferred from the change of the upstream RNA-binding regions.