In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state

It was shown decades ago that purified 30S ribosome subunits readily interconvert between “active” and “inactive” conformations in a switch that involves changes in the functionally important neck and decoding regions. However, the physiological significance of this conformational change had remained unknown. In exponentially growing Escherichia coli cells, RNA SHAPE probing revealed that 16S rRNA largely adopts the inactive conformation in stably assembled, mature 30S subunits and the active conformation in translating (70S) ribosomes. Inactive 30S subunits bind mRNA as efficiently as active subunits but initiate translation more slowly. Mutations that inhibited interconversion between states compromised translation in vivo. Binding by the small antibiotic paromomycin induced the inactive-to-active conversion, consistent with a low-energy barrier between the two states. Despite the small energetic barrier between states, but consistent with slow translation initiation and a functional role in vivo, interconversion involved large-scale changes in structure in the neck region that likely propagate across the 30S body via helix 44. These findings suggest the inactive state is a biologically relevant alternate conformation that regulates ribosome function as a conformational switch.Forty-five years ago, Zamir, Elson, and their colleagues reported that purified 30S subunits of the ribosome undergo a readily reversible conformational change between “active” and “inactive” states and proposed that this conformational rearrangement might mimic a natural process (1). Noller and coworkers used chemical probing to show that this conformational change occurs in the neck and decoding center regions of the 16S ribosomal RNA (rRNA) and has “the appearance of a reciprocal interconversion between two differently structured states” (2). Recent structural analyses indicate that the protein-free 16S rRNA adopts alternative base-paired conformations in the neck region that are conserved among diverse eubacterial and archeal organisms (3). The ability to sample multiple conformations in this region is also conserved in eukaryotes (4). The original studies on the inactive and active states noted that probing ribosomes in cells might allow the biological roles of these states to be established (1, 2). Here we make use of recent innovations in in-cell RNA SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) probing (5) to interrogate the structure of 16S rRNA in free 30S subunits, in actively translating ribosomes, and in mutant ribosomes in exponentially growing Escherichia coli.