Shin, Y. | Search bioRxiv

bioRxiv2019

Nucleated transcriptional condensates amplify gene expression

Liquid-liquid phase separation is thought to underly gene transcription, through the condensation of the large-scale nucleolus, or in smaller assemblies known as transcriptional hubs or condensates. However, phase separation has not yet been directly linked with transcriptional output, and our biophysical understanding of transcription dynamics is poor. Here, we utilize an optogenetic approach to control condensation of key FET-family transcriptional regulators, particularly TAF15. We show that amino acid sequence-dependent phase separation of TAF15 is enhanced significantly due to strong nuclear interactions with the C-terminal domain (CTD) of RNA Pol II. Nascent CTD clusters at primed genomic loci lower the energetic barrier for nucleation of TAF15 condensates, which in turn further recruit RNA Pol II to drive transcriptional output. These results suggest a model in which positive feedback between key transcriptional components drives intermittent dynamics of localized phase separation, to amplify gene expression.

biophysics↗

bioRxiv2019

Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains 8 poly-G RNA bases synthesized using 3 C bases in the DNA as a template and extends RNA without displacement of the promoter recognition {sigma} factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak trigger freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Structures and biochemical assays revealed how the RNA transcript from the pyrG promoter is guided toward the Rifampin-binding pocket then the main channel of RNA polymerase and provided insight into RNA slippage during reiterative transcription of the pyrG promoter. Lastly, we determined a structure of a reiterative transcription complex at the pyrBI promoter and revealed an alternative mechanism of RNA slippage and extension requiring the{sigma} dissociation from the core enzyme.\n\nSIGNIFICANCE STATEMENTRNA polymerase synthesizes multiple bases of RNA using a single base of the template DNA due to slippage between RNA transcript and template DNA. This noncanonical RNA synthesis is called \"reiterative transcription,\" playing several regulatory roles cellular organisms and viruses. In this study, we determined a series of X-ray crystal structures of a bacterial RNA polymerase engaged in reiterative transcription and characterized a role of template DNA during reiterative transcription by biochemical assays. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. We also provide insights into the regulation of gene expression using two alternative ways of reiterative transcription.

molecular biology↗