Graduate Thesis Or Dissertation
 

Accessibility of highly conserved RNA sequences of Escherichia coli 70S ribosomes and implications for developing antibacterial agents

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  • Escherichia coli 16S-like ribosomal RNAs from eubacterial, archaebacterial and eukaryotic organisms have several universally conserved sequence zones which are proposed to be single-stranded. A clear function for all of these regions has not yet been defined. To evaluate the roles of these sequence zones in protein synthesis, knowledge of their disposition in small ribosomal subunits and in intact ribosomes is essential. Here I report the results of experiments which were undertaken to quantitate the extent to which these sequences are single stranded and exposed in 30S subunits and 70S ribosomes from Escherichia coli D-10 cells. The approach was based upon the protection from nuclease S1 digestion of short synthetic DNA oligomers hybridized to target sequences. In Escherichia coli 16S rRNA nucleotides 520-530, 1396-1404, 1493-1504 and 1533-1542 were all found to be single stranded and stoichiometrically unrestricted for hybridization to complementary short (9-11 nucleotide) DNA oligomers. The disposition of the sequences in 30S subunits and 70S ribosomes varied with each position. To test the hypothesis that blockage of the normally open conserved sequences were essential for inhibition of protein synthesis, a set of novel synthetic short DNA analogs (3-6 nucleotides) and normal DNA (10 and 11 nucleotides) complementary to the regions 520-530 and between nucleotides 1533-1542 in 16S rRNA were tested for their ability to inhibit protein synthesis. The inhibitory activity of the DNAs were assessed in vitro and in vivo. Oligonucleotide analogs centered about position 1536-1539 inhibited protein synthesis in vitro and also inhibited colony formation by permeable and nonpermeable E. coli cells.
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