Formation of synthetic RNP granules using engineered phage-coat-protein -RNA complexes

Observations of RNA-protein (RNP) granules within living cells have generated important insights on intracellular processes in recent years. Here, we demonstrate that PP7-coat protein (PCP) and Qβ-coat protein (QCP) co-expressed with synthetic long non-coding RNA (slncRNA) encoded with multiple CP binding sites self-assemble into phase-separated synthetic RNP (SRNP) granules within E. coli cells and in vitro. SNRP granules exhibit hallmarks of RNP granule formation including phase separation from a semi-dilute liquid in vitro, and retention of structure after cell lysis. In vivo real-time tracking of SRNP granules reveals that they are found in cell poles of exponentially growing cells, and that their structure exhibits gel-like properties by the slow stochastic insertion and shedding of CP-slncRNA complexes. We propose a transcriptional model that takes into account the cytosolic structure of E. coli (i.e., the cytosol is divided into a biomolecularly dense domain in the nucleoid-dominated region and a dilute liquid domain in the polar regions), and use it to provide a theoretical framework for explaining the increase in intracellular CP-titer as a function slncRNA structure, and the appearance of SRNP granules in non-polar regions of stationary phase cells. Consequently, the SRNP-granule data and resultant model has implications for various bacterial transcriptional and translational processes, and provides an alternative explanation for the super-Poisson kinetics attributed to transcriptional bursts.