Single-Molecule Assay Development for Studying Human RNA Polymerase II Promoter-Proximal Pausing

Abstract

Promoter-proximal RNA Polymerase II (Pol-II) pausing has been shown to play a significant role in transcription regulation of elongating Pol-II complexes in a large number of metazoan and mammalian genes (1). The traditional understanding of transcription regulation in mammals involved controlling Pol-II recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. Most works investigating promoter-proximal PolII pausing have employed chromatin immunoprecipitation followed by sequencing to determine Pol-II localization or in vitro transcriptional assays using nuclear extracts analyzed with radioactive gel electrophoresis. In order to gain greater mechanistic insight into the regulation of promoter-proximal Pol-II pausing, we use single molecule ALEX spectroscopy to monitor RNA transcripts production as function of composition and order of addition of transcription factors to an in vitro reconstituted human Pol-II system. The RNA transcripts are detected by complementary doubly dye-labeled single-stranded DNA (ssDNA) probes. The human gene HSPA1B for heat shock protein 70 (Hsp70) is used as a model system due to its extensive characterization in drosophila. Our approach provides a rapid, sensitive and robust avenue for screening protein factors regulating promoter-proximal Pol-II pausing.1. H. Kwak, J. T. Lis, Control of transcriptional elongation. Annu. Rev. Genet. 47, 483-508 (2013).