Ubiquitin and ubiquitin-like (Ub/Ubl) systems are attractive targets for improving public health including the development of new therapies to overcome viral infections, cancer, neurodegeneration and other human disorders and diseases. The ‘dual function’ Ubl systems are a variant of the canonical pathways in that the Ubls are used in both Ubl protein ligation and as sulfur carriers to form 2-thiolated wobble uridine (s2U34) tRNA and molybdenum cofactor (Moco). The dual function Ubl systems are thus versatile and found to be evolutionarily conserved from archaea to humans. While relying upon an E1 for activation, the dual function Ubls do not use conserved E2 conjugating or E3 ligase enzymes for protein modification. Rhodanese domains (RHDs) serve as the apparent E2 modules of these systems and may determine whether the Ubl is used in Ubl ligation or sulfur mobilization. Nevertheless, sound evidence for the role of the RHD as an E2 analog is lacking, and its interface with the E1 and Ubl is unclear. Moreover, while components of the dual function Ubl system are required for the 2-thiolation of U34 tRNA, other factors may influence the pools of this modified tRNA to ensure proper translation efficiency and cellular homeostasis. Included among these other factors are enzymes that modify the 5-position of U34 tRNA, hydrolases predicted to alter the levels of a 2-thiolation U34 tRNA intermediate, and environmental conditions such as nutrient deprivation, heat stress and oxidation. However, the role these other factors may have in regulating s2U34 tRNA levels and the coordination of this regulation with the post-translational modification of proteins are not well understood. Furthermore, while dual function Ubl systems modify proteins including those associated with viral infections and chemotherapy resistance, knowledge of the biological roles and diversity of these modifications is limited compared to other Ub/Ubl systems. Our long-term goal is to advance understanding of dual function Ubl systems and translate this knowledge to applications of relevance to human health. Our central hypothesis is that dual function Ubl systems are important to cell function as they coordinate: i) sulfur mobilization pathways associated with translation efficiency (s2U34 tRNA) and metabolism (Moco) with ii) Ubl ligations that target proteins for destruction by proteasomes, reversible inactivation, and/or altered associations in protein-protein interaction (PPI) networks. In aim 1, we will determine how RHDs interface the E1 and Ubls to mediate and potentially regulate the dual function Ubl pathway. In aim 2, we will assess whether the 2-thiolation status of wobble uridine tRNAs is regulated, what these regulators are, and whether these regulators are correlated with the post-translational modification of proteins. In aim 3, we will determine how the PPI network of Cdc48a, RecJ3, RecJ4 and RNase J associates with proteins tagged with dual function Ubl modifiers to inf...