Graduate Thesis Or Dissertation
 

Modulation of translational fidelity by small subunit ribosoma RNA from Escherichia coli

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  • Ribosomes are ribonucleoprotein particles that are essential for the process of protein synthesis in all living systems. Escherichia coli, eubacterial organisms, have ribosomes which display a 70S sedimentation coefficient. They are formed by the association of two ribonucleoprotein subunits which sediment at 50S and 30S. The 30S subunit is a construct made of 21 different proteins and 16S ribosomal RNA. The 3'-terminus of this rRNA was the focus of the research recounted in this dissertation. This tract of 10 nucleotides in rRNA is absolutely conserved in eubacterial organisms and is asserted to be crucial for proper initiation of protein synthesis in eubacteria. Here I report the results of experiments done with 16S rRNA missing a part of the conserved 3'-terminus. These experiments were undertaken to provide a refinement of our understanding of the requirement for and function of this conserved segment of 16S rRNA. The deletions were made directly within mature 16S rRNA using RNase H and a 10 nucleotide synthetic DNA complementary to the 3'-terminus of 16S rRNA. The synthetic DNA was hybridized to 16S rRNA and then treated with RNase H. RNase H will only attack RNA when it is basepaired with DNA. This permits site-directed mutagenisis on the mature RNA. This deletion strategy efficiently yielded a 3'-terminal nucleotide deletion in E. coli 16S rRNA. This 3'-terminal deletion did not impair in vitro 30S subunit assembly. Therefore, the conservation of the sequence is not necessary for ribosome assembly. To investigate the functional properties of the modified particles an in vitro protein translation system primed with a natural mRNA was employed. The mRNA was viral MS2 messenger. Modified particles did translate the MS2 mRNA but the fidelity with which the translation occurred was diminished.
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  • File scanned at 300 ppi (Monochrome, 256 Grayscale) using Capture Perfect 3.0 on a Canon DR-9050C in PDF format. CVista PdfCompressor 4.0 was used for pdf compression and textual OCR.
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