Tuesday, April 1, 2025
4/1/2025
Mechanisms of Molecular Motors in Transcription and Protein Homeostasis - Project Summary/Abstract Motor proteins harness the energy of nucleotide binding and hydrolysis to power replication, transcription, translation, protein turnover, and much more. To fully understand molecular motors there is a need, not only for the atomic detail of the structure, but also a knowledge of the energetics and kinetics of the reactions catalyzed. Our research seeks to quantify the mechanisms of the reactions catalyzed by motor proteins in transcription and protein homeostasis. Advanced human aging results in a decline of protein homeostasis networks. This decline leads to misfolded and aggregated proteins that result in many human diseases including some cancers, type II diabetes, and neurodegenerative diseases. Protein homeostasis networks are underpinned by motor proteins from the AAA+ superfamily (ATPases Associated with various cellular Activities). We are undertaking quantitative studies of the mechanisms of enzyme catalyzed protein unfolding and translocation of three representative Class 1 AAA+ molecular motors, S. cerevisiae Hsp104, E. coli ClpB, and E. coli ClpA. Recently, there has been a substantial increase in the number of structures of AAA+ motors. However, there remains a gap between our knowledge of the three-dimensional static structure and functional mechanistic studies of these motors. Using transient state kinetics and single molecule approaches, we will fill this gap by quantitatively defining the elementary rate limiting steps for enzyme-catalyzed protein unfolding by the protein disaggregating machines, Hsp104, and ClpB, compared to ClpA, the motor component of an ATP dependent protease. This will include an interrogation of the coupling of ATP to protein unfolding and translocation as well as the impact of substrate stability on the mechanisms of unfolding. We seek to answer the long-standing question of how the kinetic mechanisms are modulated by co-chaperones? In contrast to Archaea and Bacteria that employ a single DNA- Dependent RNA polymerase (RNAP) to transcribe all genes, in Eukarya, different RNA polymerases specialize in transcription of subsets of the cellular transcriptome. All eukaryotic cells express at least three nuclear RNA polymerases: Pol I synthesizes most ribosomal RNA (rRNA); Pol II synthesizes messenger RNA (mRNA) and most regulatory RNAs; and Pol III, synthesizes transfer RNA (tRNA) and the 5S rRNA. This “division of labor” between the Pols has been known for many years, yet the functional divergence between the three eukaryotic polymerases remains poorly understood. We have embarked on a quantitative mechanistic examination and comparison of the three eukaryotic polymerases with the objective of answering the following questions: What are the unique mechanistic differences between the three Pols that make them specialists? What is the intrinsic fidelity for the three Pols? Does nuclease activity quantifiably increase fidelity and, thereby, provide proofreading activity? Does pyrophosphorolysis quantifiably increase fidelity through kinetic proofreading?
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