Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Medicinal Chemistry

First Advisor

Jason Rife

Abstract

Ribosomes form the core of the protein biosynthesis machinery and are essential to life. Ribosome biogenesis is a complex cellular process involving transcription of rRNA, pre-rRNA processing, rRNA modification and simultaneous assembly of ribosomal proteins. RNA nucleotide modification is observed in all domains of life. While there is enormous conservation of ribosome structure, very few post-transcriptional rRNA modifications have been conserved throughout evolution. A notable example of such rare conservation is the dimethylation of two adjacent adenosines in the 3’-terminal helix, a highly conserved region of the small subunit rRNA. Enzymes that carry out these dimethylations are equally conserved and are collectively known as the KsgA/Dim1 family of methyltransferases. The first member of the family, KsgA, was identified in E. coli as the determinant for resistance to the aminoglycoside antibiotic Kasugamycin. Orthologs have since been described in organisms of wide spread evolutionary origins as well as in eukaryotic cellular organelles, thus underscoring the unprecedented conservation of this family of enzymes and the resultant rRNA modification. The higher evolutionary orthologs of KsgA have adopted secondary roles in ribosome biogenesis in addition to their dimethyltransferase role. The eukaryotic ortholog, Dim1, is essential for proper processing of the primary rRNA transcript. Recently, KsgA has been speculated to function as a late stage ribosome biogenesis factor and a ΔksgA genotype in E. coli has been linked to cold sensitivity and altered ribosomal profiles. This report focuses on the biochemical characterization of KsgA and its interaction with the 30S subunit. We have established the salt conditions required for optimal KsgA methyltransferase activity while confirming that KsgA recognizes a translationally inactive conformation of 30S subunit in vitro. Our study of the functional conservation of KsgA/Dim1 enzymes in the bacterial system revealed that KsgA and the evolutionarily higher orthologs could recognize a common ribosomal substrate. This indicates that the recognition elements of both, the protein and the small subunit, have remained largely unchanged during the course of evolution. Finally, based on our site directed mutagenesis and biochemical studies, we report that KsgA binds to structural components of 16S rRNA other than the helix containing the target nucleosides.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2009

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