O-GlcNAc Transferase Activity is Essential for RNA Pol II Pausing in a Human Cell-Free Transcription System
AbstractRNA polymerase II pausing is the major regulatory point in transcription in higher eukaryotes. Despite considerable knowledge of the general transcriptional machinery that are required to recruit RNA pol II to a promoter, much less is known how a paused RNA pol II is established and its release regulated, and the entirety of the machinery is likely not known. In part, this is due to the absence of an appropriate biochemical system that functionally recapitulates RNA pol II pausing and elongation and with which the pausing machinery can be identified. We describe herein a cell-free system (CFS) derived from HeLa cells that recapitulates pausing and elongation events known to occur in vivo. We have used this system to show that O-GlcNAc transferase (OGT) activity is required to establish a paused pol II, without which RNA pol II does not pause and instead enters productive elongation. Coupled with previous observations we show that both O-GlcNAc addition and removal are functionally required for pausing and elongation, respectively. Furthermore, the CFS offers significant inroads into understanding RNA pol II pausing and its regulation.