Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.