RNA helicases are a class of enzymes that modulate RNA structure in living cells, functioning in every aspect of RNA biology from transcription to decay. However, the precise biological function of the vast majority of the ~40 eukaryotic RNA helicases is largely unknown. In the previous funding cycle, we provided one of the first identifications of in vivo enzymatic helicase targets for a DEAD-box helicase to date. Moreover, we uncovered a novel role for RNA structure remodeling in transcription termination of RNA polymerase II in S. cerevisiae. This activity is regulated in response to environmental cues to control the expression of metabolic genes, a role that we found is conserved in human cells with the mammalian DEAD-box helicase DDX5. Strikingly, OBP2- dependent non-coding transcripts termed long non-coding RNAs (lncRNAs) form RNA-DNA hybrid structures or R-loops upon inactivation of Dbp2 that function in gene regulation. However, the mechanism(s) linking these multiple observations remains unknown. Filling this gap is of key importance because misregulation of numerous RNA helicases, including DDX5, results in disease states such as neurological disorders and cancer. Major remaining questions are: 1. How does Dbp2-dependent RNA structure impact termination? 2. What is the relationship between R-loop suppression and Dbp2? 3. Does mammalian Dbp2, termed DDX5, function in termination regulation through RNA remodeling? Our central hypothesis is that Dbp2 and DDX5 are members of a novel class of epigenetic regulators that unwind RNA structures in nascent RNAs to control transcription termination and R-loop formation in response to cellular energy status. We propose to test this hypothesis with three, focused Specific Aims, which integrate newly established and innovative strategies with proven experimental techniques. In Aim 1, we will define how RNA structure impacts transcriptional termination. In Aim 2, we will determine the mechanistic relationship between Dbp2 and R-loop suppression. In Aim 3, we will identify the enzymatic targets of DDX5 in human cells and connection to transcription termination. This research is relevant to multiple aspects of RNA biology, gene regulation, and human disease.