Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Gloria M. Culver
The aim of this study is to dissect molecular events that occur during the assembly of 30S subunit of the E. coli ribosome. The 30S ribosomal subunit is made up of 16S ribosomal RNA (rRNA) and 21 proteins (S1--S21). Crystal structure of the 30S subunit has answered longstanding questions on the three-dimensional organization of its constituents. However, this view does not expose the nature of interesting cooperative movements and conformational changes that occur during the formation of this macromolecular complex. Hence, biochemical and genetic efforts are required to understand these rearrangements.;In this study, we have employed directed hydroxyl radical probing and used one of the ribosomal proteins (r-proteins) S15 as the probe to identify conformational changes that occur in 16S rRNA during different stages of assembly. S15 is one of the proteins that directly interact with the 16S rRNA and it governs the binding of four other proteins during 30S subunit formation. S15 has been converted into a probe by substituting cysteines at unique positions of the protein and attaching Fe(II) to the cysteines to form Fe(II) tethered S15 proteins (Fe(II)-S15). Hydroxyl radicals can be generated from these tethered sites by Fenton chemistry. The "directed" hydroxyl radicals so produced cleave RNA elements that are in proximity to the tethered sites and these cleavage sites are mapped by primer extension.;Using the recombinant in vitro reconstitution system and this directed hydroxyl radical probing approach, we have studied the folding of 16S rRNA proximal to S15 during the course of 30S subunit assembly. These studies have revealed protein-dependent conformational changes that occur in RNA environment of S15. Our work suggests that binding of r-proteins can result in changes that are quite remote from their primary binding site and that assembly of different domains can influence one another.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Jagannathan, Indumathi, "Assembly of the 30S ribosomal subunit: S15 as the probe " (2004). Retrospective Theses and Dissertations. 949.