Mechanisms of transcription pausing and fidelity in prokaryotes and eukaryotes

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

In this project, my lab published 2 research papers (PNAS and Nature Communications), and a review article in Critical Reviews in Biochemistry and Molecular Biology. We also have one collaborative manuscript published in 2021 in eLife that describes the role of elongation factor NusG in transcription termination in B. subtilis. We completed developed a new powerful NGS method involving a combination of ribonuclease-coupled NET-seq (RNET-seq) and DNA-seq that allows genome-wide mapping of pause sites for E. coli, B. subtilis and human RNA polymerase II (Pol II) with a single base pair resolution. This methodology allowed us to determine translocation register of RNA polymerase at each pause site and identify the underlying causes of pausing. This method enabled direct examination of the role of ALL general and specialized transcription elongation and termination factors NusA, NusG, NusB, Rho, RfaH, SuhB, RpoD (sigma70), GreA, GreB, mfd, and UvrD in pausing and promoter escape in vivo. This goal was achieved by applying RNET-seq/DNA-seq to the genetically modified bacterial strains carrying the systematic gene knockouts and conditional knockdowns (using inducible dCas9 transcription roadblocks) of the corresponding target gene followed by biochemical evaluation of the mechanism of pausing at the representative pause sites in vitro. By using RNET-seq, we were first to demonstrate the crucial role of sigma70 subunit and Gre transcript cleavage factors in regulation of promoter escape at a striking large number of E. coli promoters primarily involved in regulation of stress response and cellular responses to changing environmental cues (Nature Communications). We also elucidated the role of elongation factors NusA and NusG in regulation of transcription pausing and termination in E. coli and B. subtilis (PNAS, eLife, and manuscript in preparation). Transcription fidelity: In our approach to study transcription fidelity, we combine the efficiency and high-sensitivity of the novel cre/lox-based genetic assay, developed for the yeast and E. coli cells in our group, as a team effort with three other RBL groups, with the power and precision of biochemical analysis of RNAP mutants, transcription factors and reaction conditions promoting transcription errors in these organisms. We also study in vitro the mechanisms of transcription fidelity in higher eukaryotes. For genetic assay, we create site-directed chromosomal mutants and gene constructs using advanced high-precision methods of gene manipulations based on recombineering developed in collaboration with Don Court's group. Application of the modern NGS RNA sequencing techniques brings our analysis of transcription errors to a genome-wide scale. The use of four biological models, namely E. coli, B. subtilis, S. cerevisiae and human triple negative breast cancer (TNBC) cells under normal and stress conditions, allows us to do the cross-species and cross-kingdom study of transcription fidelity helping to unravel conserved and unique features of strategies that various organisms implement to control transcription fidelity. We believe that the experimental strategy and techniques that we use in the current work lay a solid foundation for our future studies on transcription fidelity. In 2020-21, my lab continued active intervention to the new area of transcription research targeting the role of pausing of human Pol II in transcription-coupled events of 5' RNA capping, co-transcriptional splicing and transcription termination in triple negative breast cancer cells (TNBCs). This study will help us in identifying the novel splicing isoforms genome-wide, which are deregulated in TNBC cells. Since hypoxic stress is an emerging hallmark of TNBCs, we aim to determine how the RNA capping, co-transcriptional splicing and transcription termination downstream from polyA sites are deregulated during hypoxic stress. Our preliminary data strongly indicate that co-transcriptional splicing and transcription termination downstream from the polyA sites are significantly altered in TNBC cells compared to their non-TNBC counterparts, including these events in hypoxia-induced genes.