Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—implications for the study of ribosome dynamics

Abstract

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg²⁺ is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg²⁺ concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg²⁺ that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg²⁺ concentrations. In low Mg²⁺ concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.