Single molecule studies of transcription elongation factor dynamics during RNA polymerase II transcription
Creators: name: Herlambang, Karina Saskia
Publisher: name: Brandeis University
Year: 2024
Subjects: CoSMoS
RNA polymerase
Transcription factor
yeast transcription

description: Gene expression via transcription is a highly regulated process across all organisms. Transcription can be divided into three phases: initiation, elongation, and termination. For each stage a plethora of accessory proteins are required to assist RNA polymerase. While a lot of these transcription factors have been identified, many of them are still mysterious in what they do. Therefore, one of the biggest challenges in the field is to understand how all these factors work in a coordinated way to enable transcription. From in vivo studies, we know a lot of the events during transcription happen in the order of seconds. Therefore, most bulk assays are not able to detect any unstable intermediates or heterogeneity in the pathway because they are essentially only measuring the averaged behavior of the species present. Colocalization single-molecule spectroscopy (CoSMoS) enables simultaneous observation in real time of individual components of transcription in a physiologically faithful context. This technique has provided us with new details into the pathway, kinetics, and conformational dynamics of complex biological processes including transcription in bacteria and eukaryotes.In chapter 2, I report a bulk measurement of sense and antisense transcripts produced from linear and circular DNA templates using RT-qPCR and capillary electrophoresis to support the post-termination secondary initiation pathway that was previously observed in a single molecule experiment. The result supports the idea that following termination RNAP lingers on the DNA and can reassociate with σ70 to initiate another round of transcription in the opposite direction, producing antisense transcripts. Such behavior could be used by bacteria as a rapid negative feedback or gene coupling mechanism.
description: In chapter 3, I investigate the step(s) at which recombinant TFIIS (rTFIIS) stimulates transcription. I add rTFIIS to kinetically characterized yeast nuclear extract containing either labeled initiation (TFIIE and TFIIH) or elongation factor (Spt5) and RNA polymerase II (Pol II) in CoSMoS experiment. We show that rTFIIS accelerates the recruitment of Pol II to the promoter and binding of Spt5 to Pol II. In contrast, rTFIIS does not affect TFIIE binding kinetics and decreases Hek2 binding frequencies. Despite variability between experimental replicates, my preliminary results suggest that rTFIIS stimulates the formation of preinitiation complex (PIC) at the promoter and stabilizes against elongation complex dissociation.
description: In chapter 4, I studied the dynamics of other transcription elongation factors including Elf1, Bye1, and Paf1 in CoSMoS experiments. Unfortunately, either nuclear extracts with tagged factors were not transcriptionally active or the labeled factors were not observed to interact with Pol II in single molecule experiment. The complexity of the transcription elongation complex may pose a challenge to tagging the proteins without compromising their binding to other accessory proteins, or transcriptional activity. However, the results provided here may be used as a framework and guidance for future single molecule studies on RNA Polymerase (Pol) II transcription elongation factors.