Dissecting the relationships between RNA structure and function in E. coli small ribosomal subunits

Abstract

Thesis (Ph. D.)--University of Rochester. Dept. of Biology, 2010.Ribonucleic acids (RNAs) perform widely different biological functions ranging from regulation of gene expression to catalysis. Their functions are often tightly correlated with their structure. To explore the relationship between RNA structure and function, Escherichia coli (E. coli) small ribosomal subunit was used as a model system and its biogenesis was investigated using two different approaches. Firstly, we studied a RNA modification system which involves dimethylation of two universally conserved adjacent adenosines in the ultimate helix of the small subunit ribosomal RNAs (rRNA) and the KsgA (E. coli) methyltransferase family, enzyme responsible for these two dimethylations, which also appears to be universally conserved. We localized the position of KsgA relative to 30S subunits using directed hydroxyl radical probing and demonstrated that binding of KsgA and translation initiation factor 3 (IF3) are generally mutually exclusive. Our work strongly suggested that this modification system plays a critical role in differentiating fully assembled, functional subunits from those that are still involved in the biogenesis pathway. Secondly, we devised a modification interference approach to investigate nucleotides within 16S rRNA that are potentially important for the assembly of functional 30S subunits. We found that the majority of these nucleotides are located in the head and interdomain junction of the 30S subunit indicating that the different domains of the 30S subunit assemble differentially and that the assembly of the 3’ domain and interdomain junction are more restricted than assembly of the other domains. Further in vivo investigation of individual residues identified in vitro delineated the importance of these residues in ribosome biogenesis. Overall, this dissertation has demonstrated the impact of 16S rRNA structural changes on biogenesis of the small ribosomal subunit and has enabled deeper understanding of ribosome assembly