Date of Award

4-1-1995

Document Type

Thesis

Degree Name

Master of Arts (MA)

Department

Biology

First Advisor

Dr. William Tapprich

Abstract

Several phylogenetically conserved single-stranded regions within the secondary structure of 16S ribosomal RNA exhibit essential functional roles in protein synthesis. These dynamic residues adopt several three-dimensional conformations, as they interact with the 50S subunit another intercellular components. The central domain region of 16S ribosomal RNA plays a major role in this process by providing several single-stranded loop regions. The largest and most active of these loop regions surrounds position 790. The 790 loop is universally conserved amongst all sequenced species in specific residues have proven to be highly active. For example, a large body of evidence supports a direct role for the 790 loop in the initiation of protein synthesis. In this study, position 790 was investigated for its role in subunit association. Single base subunits changing 790A to C, G and U were constructed in the Escherichia coli rrnB operon on a multi-copy plasmid. The effects of the mutations on subunit association was monitored by both in vitro and in vivo assays. Cells containing 790C, 790 G, and 790U all displayed an increase in generation time. In fact, the 790G substitution resulted in a mutant ribosome that were unable to support cell growth. In vivo subunit association was dramatically reduced for cells containing 790C and 790U. This was confirmed when in vitro subunit association assays showed distinct depletions in 70S ribosomal pools. In vitro subunit association identified a pronounced increase in the affinity between subunits with the 790G mutation. All evidence suggests that position 790 plays a vitally important role in subunit association and sequence initiation of protein synthesis.

Comments

A Thesis Presented to the Department of Biology and the Faculty of the Graduate College University of Nebraska In Partial Fulfillment of the Requirements for the Degree Master of Arts University of Nebraska at Omaha. Copyright 1995 William R. Staplin.

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