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Papers In Press, published online ahead of print October 30, 2000
J. Biol. Chem, 10.1074/jbc.M008616200
Submitted on September 20, 2000
Revised on October 30, 2000
Accepted on October 27, 2000

Two-site Contact of Elongating Transcripts to Phage T7 RNA Polymerase at C-terminal Regions

Haihong Shen and Changwon Kang

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701

Corresponding Author: ckang{at}mail.kaist.ac.kr

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 incorporated at the 22nd, 24th, 32nd, and 38th residues separately. 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. 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. Linkage of the 3?-end residue is mapped to the 636Thr-666Met region, which contains nucleotide-binding sites. The ?9 residue is cross-linked to the 724Ala-750Met region rather than an N-terminal region. These two contacts are maintained throughout the elongation complexes, and reveal a route of nascent RNA through the T7 RNA polymerase in elongation complexes.


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