Functions of the conserved ribosome-bound protein Lso2 in translation and physiology

Abstract

The ribosome is a highly conserved macromolecular machine that carries out translation, the synthesis of proteins from mRNAs, in all domains of life. The core ribosome interacts with dozens of general translation factors that ensure accurate and efficient progression through the translation cycle. Their detailed characterization has significantly advanced our understanding of protein synthesis. However, a growing number of ribosome-associated proteins have also been discovered whose functions are less well understood. In Chapter 1, I will overview the translation cycle and describe how it is affected by nutrient availability, with a focus on functions of starvation-induced proteins that directly bind the ribosome. I will also discuss discovery approaches for expanding the study of ribosome-associated proteins. In Chapter 2, I will present the discovery and characterization of Lso2 as a conserved ribosome-bound protein required for translational recovery in budding yeast. Using quantitative mass spectrometry, we found this protein to be ribosome-associated during glucose-starved and replete growth, with moderate enrichment on translating ribosomes during starvation. Saccharomyces cerevisiae lacking Lso2 accumulate monoribosomes that are not translating normally following a shift from stationary phase to rich medium. To understand the basis of this phenotype, we used genome-wide RNA crosslinking and sequencing to determine that Lso2 binds near the A site of the ribosome tRNA channel, in a region that overlaps with the GTPase activating center, and that Lso2 also interacts with a broad spectrum of tRNAs. Consistently, Lso2 binding in the tRNA channel stabilizes ribosomal subunit association in vitro. These data, together with evidence that the accumulated ribosomes in Iso2 nulls are devoid of obvious barriers to initiation, lead to a model in which Lso2 promotes productive elongation. Finally, I show that the ribosome binding activity of Lso2 is conserved in its human ortholog, suggesting a broad importance of its molecular function. In Chapter 3, I will elaborate on the model of a function for Lso2 in elongation, propose alternative models to rationalize its effects on translation, and describe experiments for testing them. I will also describe the implications of this protein for our understanding of translation in different physiological states.