The focus of this proposal is on the evolution of #pausing#, one of the regulatory stages during transcription by RNA Polymerase II (Pol II) that has so far been identified only in animals. Transcription is regulated at early stages in all eukaryotic organisms, including opening chromatin at the proximal promoter and forming the pre-initiation complex. In Drosophila and mammals, Pol II pauses near the transcription start site and awaits a signal to #release# into productive elongation. Our work has demonstrated that the rate at which Pol II is released from a paused state is an active target of gene regulation by transcription factors. Therefore pausing adds a layer of regulatory complexity that has not been reported outside of animal organisms.

Here we propose to investigate the evolution of pausing in organisms at the base of the animal phylogeny. We present a specific hypothesis for how pausing evolved by collapsing an ancestral step necessary for transcription in all eukaryotes to a focal pause always occurring in the same location along the DNA sequence. By having a focal pause that occurs in the same location, transcription factors were able to efficiently catalyze the release of paused Pol II. This allowed release from pause to become a step during mRNA production that is actively regulated by cells. Our proposal to investigate this hypothesis will provide mechanistic insights into how pausing evolved in eukaryotic species, revealing how evolution added a step in complexity to an essential biological process.

Our project will advance the goals of the NASA Exobiology program by determining when pausing evolved and by providing the mechanistic details of how this process developed into an active target of regulation observed in extant animals. Thus, our proposal addresses the biological factors essential to multicellular life and the evolution of multicellularity on Earth.