How does a universally conserved GTPase Drg promote efficient translation in the cell?

University Of Illinois At Urbana-Champaign, Urbana IL

Investigators

Abstract

Ribosomes synthesize every protein in the cell. In the crowded cellular environment, translating ribosomes pause, stall or even collide, but how these “troubled” ribosomes recover and resume protein synthesis is unclear. Furthermore, little is known about how the cell distinguishes programmed, physiologically important pauses for proper protein folding and targeting versus problematic, even pathological stalls due to translation errors. To uncover the responsible cellular pathways and molecular interactions, this project will investigate how the conserved, developmentally regulated GTP-binding protein Drg promotes protein synthesis when translating ribosomes slow down. The outcomes of this investigation will provide new insights and establish new principles for regulation of gene expression. In addition, the educational goals of the project include new curriculum development and integration of research and education with an emphasis on creating more opportunities for studnets in STEM. As an ancient GTPase, Drg protein has co-evolved with the ribosome, and recent findings indicate that it can serve as a new general translation factor. To explore its role in translation, two complementary aims will be pursued in this study. Aim 1 seeks mechanistic understanding of Drg-mediated functions in bacterial and eukaryotic translation through in vitro biochemical studies of fully reconstituted translation systems from E. coli and S. cerevisiae. Translation assays will be utilized to probe the conformation of the peptidyl transferase center and to measure the kinetics of peptide bond formation, which will clarify the nature and extent to which Drg affects translation when ribosomes pause. Aim 2 seeks to uncover molecular interactions important for Drg function. APEX proximity labeling will be used to determine Drg interactomes in E. coli and S. cerevisiae cells, which will be followed by mutagenesis and biochemical studies. The findings from this study will elucidate an evolutionarily conserved mechanism by which cells can regulate and fine-tune proteostasis. 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.