The overall goal of the proposed work is to understand the mechanism of assembly of the 30S ribosomal subunit. We have made a systematic set of deletions to 16S rRNA to generate fragments of the 30S subunit that correspond to domains, subdomains and assembly intermediates. Detailed thermodynamic and structural studies on these complexes will provide insights into the cooperative nature of protein binding and RNA conformational changes during 30S assembly. In the previous grant periods, we focused on the central domain of the 30S subunit, and we now extend our studies to include the 5'- and 3'-domains. In addition, we have developed a stable isotope (15N) pulse-chase assay that allows us to monitor binding of all 20 proteins to the 16S rRNA simultaneously, using a mass spectrometric analysis.
The Specific Aims for the next period are: 1) Understanding the assembly mechanism of the 30S central domain. Building upon our previous work, we will probe the role of RNA tertiary interactions in the later stages of central domain assembly. 2) Biochemical analysis of the 5'- and 3'-domains of the 30S subunit. Using a similar approach that we developed for the central domain, we will develop minimal binding sites for each 5'-and 3'-domain proteins, and probe the early stages of assembly of the 3'-domain. 3) Monitoring assembly of the 30S subunit using isotope pulse-chase mass spectrometry. Assembly of the 30S will be initiated using 15N-labeled ribosomal proteins, and then chased with unlabeled 14N proteins. After purification of the intact 30S subunits, a proteomic mass spectrometry analysis will be performed to analyze the 15N:14N ratio for each protein as a function of the assembly time. This will allow us to monitor the 30S assembly from a global perspective. ? ?
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