Tome, Jacob Michael2018-10-232019-06-042018-05-30Tome_cornellgrad_0058F_10735http://dissertations.umi.com/cornellgrad:10735bibid: 10489365https://hdl.handle.net/1813/59281To understand gene regulation, we need both targeted approaches to probe individual regulatory components, and systems level approaches to understand the functional state of cells. Presented here are several studies at different points on this functional spectrum, with a focus on the crucial regulatory step of promoter proximal pausing. RNA-protein interactions have critical roles in gene regulation. We adapted an Illumina GAIIx sequencer to make several millions of these measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its template DNA. Binding of fluorescently-labeled protein is then quantified in the sequencer. By measuring the affinity of mutagenized libraries of an RNA aptamer to NELF-E, an RNA binding subunit of the pausing factor NELF, we show that this interaction is due to a short RNA motif, but the three dimensional structure of the aptamer is critical for its high affinity. We used this aptamer as an in vivo inhibitor of the interaction between NELF-E and nascent RNA in Drosophila S2 cells. Pausing was globally reduced, but promoters with the transcription factor GAF were unchanged. Thus, the interaction between NELF-E and nascent RNA is not critical for pausing when GAF aids NELF recruitment, but is a strong component of NELF recruitment elsewhere. In higher eukaryotes, the timing and level of transcription at gene promoters by RNA Polymerase II (Pol II) is specified largely by the sum of information from the promoter itself and from distal enhancers .Transcription widely occurs at enhancers, suggesting Pol II may be a ubiquitous nexus of regulatory signaling. To explore this, we sequenced nascent RNAs at single-molecule resolution to identify Pol II initiation, capping, and pause sites. Our analyses reveal distinct sequence-specified pause classes associated with differences in RNA capping dynamics. Initiation typically occurs within large clusters, especially at gene promoters. Integrated analysis of nearby chromatin and transcription factors suggests a model of gene regulation in which Pol II initiation provides a biophysical scaffold to create and maintain regulatory domains.en-USAttribution-NoDerivatives 4.0 InternationalBiochemistryMolecular biologyGene regulationAptamerNELFPromoter proximal pausingRNA-protein interactionsTranscriptiondissertation or thesishttps://doi.org/10.7298/X4Q81B93