Manipulating archaeal systems to permit analyses of transcription elongation-termination decisions in vitro

Transcription elongation by multisubunit RNA polymerases (RNAPs) is processive, but neither uniform nor continuous. Regulatory events during elongation include pausing, backtracking, arrest, and transcription termination, and it is critical to determine whether the absence of continued synthesis is transient or permanent. Here we describe mechanisms to generate large quantities of stable archaeal elongation complexes on a solid support to permit (1) single-round transcription, (2) walking of RNAP to any defined template position, and (3) discrimination of transcripts that are associated with RNAP from those that are released to solution. This methodology is based on untagged proteins transcribing biotin- and digoxigenin-labeled DNA templates in association with paramagnetic particles.