In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Abstract

While structuring of the bacterial nucleoid by nucleoid-associated proteins (NAPs) is critical for proper chromosomal organization and compaction, DNA-dependent RNA polymerase (RNAP) must frequently interact with and overcome the barriers these NAPs impose upon transcription. One particular NAP in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS, that binds to DNA and forms nucleoprotein filaments. To specifically investigate the effect that H-NS filaments have on RNAP elongation, we developed an in vitro transcription assay to assess transcript elongation by RNAP when transcribing DNA bound by an H-NS filament. In this method, initiation and elongation by RNAP are uncoupled by initiating transcription in the presence of three rNTPs to halt elongation just downstream of the promoter. Before elongation is restarted, an H-NS filament is formed so that elongation occurs on an H-NS nucleoprotein filament template. We also describe visualization and analysis of the transcription products from the nucleoprotein template which provides insight into how H-NS and RNAP interact. This method is a starting point to determine effects of NAPs on RNAP elongation in a variety of conditions.