Mapping structural differences between 30S ribosomal subunit assembly intermediates

Under appropriate conditions, functional Escherichia coli 30S ribosomal subunits assemble in vitro from purified components. However, at low temperatures, assembly stalls, producing an intermediate ( RI) that sediments at 21S and is composed of 16S ribosomal RNA (rRNA) and a subset of ribosomal proteins (r-proteins). Incubation of RI at elevated temperatures produces a particle, RI*, of similar composition but different sedimentation coefficient (26S). Once formed, RI* rapidly associates with the remaining r-proteins to produce mature 30S subunits. To understand the nature of this transition from RI to RI*, changes in the reactivity of 16S rRNA between these two states were monitored by chemical modification and primer extension analysis. Evaluation of this data using structural and biochemical information reveals that many changes are r-protein dependent and some are clustered in functional regions, suggesting that this transition is an important step in functional 30S subunit formation.