Mediator Forms Clusters with RNA Polymerase II in Live Stem Cells

Abstract

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.