Mechanisms of Fidelity in Eukaryotic RNA Polymerases

University Of Alabama At Birmingham, Birmingham AL

Investigators

Abstract

Highly accurate copying of DNA into RNA is essential for cell growth and proliferation across all domains of life. This project will fill a fundamental gap in our understanding of the chemical reactions involved in copying the information contained within DNA into RNA with a low occurrence of errors. The project will produce a detailed description of how the enzymes involved extend the RNA chain and perform error correction. The results produced will aid the broader scientific community in understanding why higher organisms have evolved three specialized enzymes to copy specific classes of genes when lower organisms use a single enzyme to copy all genes. The project will train students and produce Ph.D.-level experts who will be sought by both the public and private sector for their ability to apply rigorous biophysical approaches. Undergraduate students will also be trained in these approaches. This exposure will allow the students to be more competitive for future careers in STEM. High-fidelity (low probability of error) gene expression is essential for organisms in all domains of life. Archaea and Bacteria employ a single RNA polymerase, RNAP, to transcribe all genes in the prokaryotic cell. In contrast, eukaryotes have at least three RNA polymerases (Pols). Pol I synthesizes ribosomal (r) RNA, Pol II synthesizes messenger RNA and most regulatory RNA, and Pol III synthesizes the 5 S rRNA and transfer RNA. It has been hypothesized that Pol II evolved from the archaeal RNAP and Pol I and III evolved from Pol II via sequence divergence of core subunits and incorporation of transcription factors as bona fide subunits. These observations suggest that the eukaryotic polymerases have evolved divergent mechanisms of transcription and transcription fidelity making them uniquely suited to transcribe their target genes. The project will interrogate the role of subunits within these multi-subunit RNA polymerases that are related to error correction. The overall goal of this work is to employ transient state kinetic approaches to inform the evolutionary divergence by quantitatively defining the kinetic mechanisms of transcription and transcription fidelity for the three Pols. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.