Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.

A major unresolved question in the mechanism of transcription initiation by the multi-subunit Escherichia coli RNA polymerase (RNAP, subunits a 2 bb ' us 70 ) is the relevance of the wide range of open complex (OC) lifetimes exhibited by different promoters. OC lifetimes for promoters lP R , T7A1, and rrnB P1 show OC lifetimes range from >10 5 s (lP R ) to less than 1 s (rrnB P1). An elaborate network of in-cleft and downstream interactions directed by the discriminator region-just upstream of the transcription start site-is involved in lifetime determination. We recently presented evidence that longer-lived, more stable OC synthesize a longer RNA-DNA hybrid before escape. Consequently, we've seen a wider length range of short (abortive) RNA from longer-lived, more stable open complexes, while few short RNA of any length are synthesized from short lived OC under conditions investigated. It is unclear if these differences in short RNA distribution are the only consequence of the large differences in OC lifetime and stability, and the roles of these short RNAs in regulation of gene expression are also largely unknown. My research focuses on in vivo effects of discriminator sequence and length variation by utilizing synthetic plasmid libraries, promoting a super folder green fluorescent protein (sfGFP) gene, and with upstream promoter sequences homologous to either lP R , T7A1, or rrnB P1. E. coli cells transformed with these libraries are measured for sfGFP expression via flow cytometry, sorted by expression level using fluorescence-activated cell sorting (FACS), and cells of each expression level queried for discriminator consensuses using Illumina sequencing. Our sequence findings, with in vitro studies using consensus sequences, will enable us to elucidate regulatory effects of the discriminator region, getting us closer to the mechanistic explanation for the large range of OC formation in bacteria.