PROJECT SUMMARY In all organisms, proteins are synthesized by ribosomes, large two-subunit enzymes that use aminoacyl-tRNA substrates to translate messenger RNA. Each ribosome is composed of several large RNA molecules (rRNAs) and more than 50 distinct proteins (r proteins), with rRNA accounting for around two-thirds of the overall mass. Seminal studies by the Nomura and Nierhaus groups showed that each ribosomal subunit can be reconstituted in vitro from its purified components, and hence the ribosome is fundamentally capable of self-assembly. However, such self-assembly is slow, inefficient, and requires non-physiological conditions. In the cell, numerous auxiliary proteins (termed assembly factors or AFs) facilitate the assembly process. Presumably, these AFs prevent and/or resolve low-energy intermediates (e.g., rRNA folding traps). But how these AFs act remains largely unknown, even in the simplest bacterial system. The long-term goal of the proposed work is to understand how rapid ribosome assembly is achieved in the cell. Aim 1. The GTPase BipA is a paralog of EF-G that plays some unclear role in 50S biogenesis. Cells lacking BipA grow poorly at low temperature and accumulate particles that represent a novel pre-50S (~40S) intermediate. The proposed work will characterize the structure of this intermediate, using chemical probing and cryo-electron microscopy approaches. The findings may define an intrinsic rRNA folding issue in 50S assembly and reveal the normal role of BipA. Aim 2. A growing body of evidence suggests that late-state 30S assembly occurs in the context of the 70S ribosome. The proposed work will investigate whether 30S biogenesis in bacteria includes a “test drive,” a translation-like cycle of quality control, as has been reported in eukaryotic cells. The findings may reveal an unappreciated functional link between ribosome assembly and translation initiation in bacteria. Aim 3. There currently exists no convenient assay to directly monitor 30S assembly in vitro. A FRET-based assay will be developed and used to investigate the roles of AFs, precursor rRNA elements, initiation components, and concurrent transcription on the rate of 30S assembly. Cellular components and/or parameters critical for rapid 30S biogenesis may be revealed and their mechanisms elucidated. Ribosomes are a main target of antibiotics, and defects in ribosome biogenesis cause many inherited human diseases (termed ribosomopathies). Insight gained by this project may ultimately lead to the development of novel antimicrobial drugs and/or treatments for one or more hereditary diseases.