A systematic analysis of ribosomal small subunit biogenesis in wild-type E. coli

Abstract

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2014.Ribonucleoproteins (RNPs) perform diverse biological functions, from catalysis to regulation of gene expression. Ribosomes are complex RNPs that synthesize proteins in all living organisms. Ribosome assembly in bacteria has been studied using in vitro reconstitution experiments for over five decades. In vitro reconstitution is not truly representative of ribosome assembly as in vivo ribosome biogenesis requires transcription, processing and modification of 5000 nucleotides of ribosomal RNA (rRNA), and is aided by several auxiliary factors, which are generally not included in in vitro reconstitution. In vivo ribosome biogenesis in bacteria is poorly understood because it is a complex, efficient and asynchronous process with only a small pool of intermediates present in wild-type cells at any given time. This thesis presents novel techniques and findings on the assembly of ribosomal small subunit (SSU) in wild-type E. coli under optimal growth conditions. To study SSU assembly in E. coli, we developed RNP affinity purification techniques to isolate and characterize in vivo formed SSU intermediates. These approaches took advantage of the regions in precursor 16S rRNA (pre-16S rRNA, leader and trailer) that are components of the pre- rRNA and SSU intermediates but are absent in mature SSUs or ribosomes. An RNA affinity tag was inserted in pre-16S rRNA at different positions between various nucleolytic cleavage sites, allowing systematic purification of different intermediates and mapping of the assembly cascade. The first precursor of 16S rRNA (17S rRNA) is the major platform for SSU biogenesis in vivo. Structural probing demonstrated that these purified 17S rRNA containing SSU intermediates had diverse architectures representing early to late stages of SSU biogenesis. These intermediates are likely incapable of translation as the regions of 16S rRNA involved in translation showed altered structure compared to the corresponding regions in mature SSUs suggesting there are checkpoints that prevent immature subunits to enter the translation cycle. The three pre-SSUs exhibited differential association of ribosomal proteins and known auxiliary factors and thus revealed multiple pathways for these processes during SSU biogenesis. Several novel and putative auxiliary factors were also identified using proteomic analysis, and preliminary characterization had demonstrated their role in SSU biogenesis. Additionally, substrates for two 16S rRNA modification enzymes were partially characterized. Our results indicate that there are multiple pathways for different biogenesis processes and SSU assembly occurs largely on 17S rRNA. Final 17S rRNA processing events happen late in the biogenesis cascade and follow multiple pathways with independent 5′ end or 3′ end maturation of 17S rRNA. These findings allow the first integration of rRNA processing events with conformational changes, rRNA modification, r-proteins association and auxiliary factor action during ribosomal SSU biogenesis in wild-type bacteria