An Investigation of Intersubunit Bridges Important for Ribosome Stability in Saccharomyces Cerevisiae

Abstract

The ribosome, a large ribonucleoprotein complex responsible for protein biosynthesis, is composed of two unequal subunits that must correctly associate for 1 proper function. X-ray crystallographic and cryo-EM studies have identified a number of specific interactions, or intersubunit bridges (ISBs ), between the large and small subunits. At least two of these ISBs have been shown to be important for ribosome stability in bacteria using a technique known as modification interference. A similar modification interference approach is presented to identify ISBs that are important for ribosome stability in yeast. Native ribosomes and ribosomal subunits were isolated from yeast cells. Sucrose density gradient ultracentrifugation under a variety of conditions was investigated in order to achieve separation and purification of individual ribosomal subunits from intact ribosomes. The determination of the optimal conditions for subunit separation and isolation is a necessary step toward identifying possible ISBs through modification int~rf erence. We hope our modification interference investigations will provide insights regarding the function and conservation of ISBs.