Mechanisms of transcription pausing and fidelity in prokaryotes and eukaryotes

Division Of Basic Sciences - Nci

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Abstract

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, Bacillus, 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.