Graduate Thesis Or Dissertation
 

Site-specific 3'-terminal deletions in Escherichia coli 16S rRNA and the effect on assembly of 30S ribosomal subunits

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  • The ribosome is a central component of the protein synthetic apparatus. It is a macromolecular complex of protein and RNA. Although much progress has been made in understanding the functional role of the proteins in this particle, little is known of the functional role which the RNA plays. Naturally occurring primary structural mutations in rRNAs have not been reported. The work described here focused on developing methodology for generating site-specific deletions in rRNA directly to explore the functional properties of the RNA. The 3'-terminus of E. coli small subunit 16S rRNA was chosen as the site to be deleted. This region of the RNA is believed to be important in the initiation of protein synthesis and could be essential for proper assembly of ribosomes. The deletions were achieved by synthesizing a DNA molecule of 10 bases in length which was complementary to the 3'-end of 16S rRNA. The DNA was hybridized to the RNA and then the RNA component of the hybrid was specifically digested with a combination of RNase H isolated from E. coli and calf thymus. This produced an equal mixture of "16S" RNAs missing either 10 or 8 terminal nucleotides. This RNA was shown to be functional in in vitro reconstitution studies indicating that this zone of the RNA is not essential for ribosome assembly.
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