Understanding 16S ribosomal RNA conformational changes during 30S subunit assembly

Abstract

The purpose of this course of study is to understand the conformational changes that occur in 16S rRNA during ribosomal assembly. The prokaryotic ribosome comprises two asymmetric subunits, the large (50S) and small (30S) subunits. The 30S subunit contains 16S rRNA and the small ribosomal proteins (S1-S21). Functional 30S ribosomal subunits can be reconstituted in vitro from purified components. Reconstitution performed at low temperatures results in a stall in 30S subunit assembly. This stall produces a Reconstitution Intermediate (RI) which sediments at 21S and contains 16S rRNA and a subset of small subunit proteins. In order for RI to be converted into functional 30S subunits, a temperature shift is first required. This shift produces a particle, RI*, with the same composition as RI, yet a dramatically different sedimentation coefficient (26S). RI* can then go on to bind the remaining small subunit proteins, ultimately resulting in functional 30S subunit formation. To better understand the nature of these assembly transitions and thus small subunit assembly, changes in reactivity of each nucleotide during the course of assembly was monitored. The changes in reactivity of nucleotides in 16S rRNA between each of the assembly species (16S, RI, RI* and 30S) were analyzed in the context of the 30S subunit structure and previous biochemical studies to better understand the relationship between ribosomal assembly and function.