Characteristic function of the conserved sequence on the sequence-specific termination by T7 RNA polymerase

Abstract

A sequence-specific termination signal of E. coli operon rrnB t1 recognized by bacteriophage RNA polymerases consists of conserved upstream-module (ATCTGTT in the non-template strand) and U-rich downstream-module. We previously elucidated the formation of paused conformation just prior to the point of RNA release. In this study, we observed sequential characteristic changes of ternary complex from more previous to the termination site. Elongation complexes of T7 RNA polymerase were stopped at desired positions by stepwise walking utilizing complex immobilization via biotin-streptavidin conjugation. The significant 9-nt upstream contact observed by photocross-link between nascent RNA and RNA polymerase in the elongation mode remarkably diminished from the third nucleotide position of the upstream-module. The release rate of the RNA in ternary complex was abruptly increased just before the conserved sequence and further increased near the point of RNA release if DNA templates contained intact upstream-module. Templates mutated termination signal showed that upstream-module appeared to contribute characteristic changes to cross-linking pattern and complex stability. At a few bp before the RNA release site, the slow intermediate complexes requiring high NTP concentration was observed. The collapse of transcription bubble was shown prior to termination. The normal transcription bubble shrank in the complex possessed intact upstream-module termination signal. The sequential changes in elongating complex before termination suggested that termination is prepared from 15bp before the RNA release site and accompanied by the various conformational changes.