ANN SCHEUNEMANN

A group of antibiotics called aminoglycosides, used mainly against gram-negative bacteria, bind to ribosomal RNA causing codon misreadings by tRNA. The aminoglycoside paromomycin interacts with a segment of the E. coli 16S rRNA Helix 44, and the binding can be characterized by an increase in temperature for the major UV-monitored thermal transition. We have initiated an investigation of a different rRNA sequence, that of 23S rRNA Helix 69. The nucleoside sequence from residue 1906 to 1924 was chemically synthesized with and without the naturally occurring modified nucleosides. This sequence was chosen because it contains three highly conserved pseudouridines (?) at positions 1911, 1915, and 1917, and because it also contains multiple adenines that create an A-platform reported to be optimal for aminoglycoside binding. Because the thermal stability of the RNA is altered by the binding of the aminoglycoside, a change in Tm of Helix 69 would be indicative of binding. The modified E. coli Helix 69 produced a definitive high temperature melting transition when in the presence of paromomycin. Titrations of the modified Helix 69 with paromomycin indicated that two-site binding affinities could be extracted to determine the dissociation constants, Kd = 2.7 and 4.9 μM. Titrations of unmodified Helix 69 also produced an increasing Tm, which could be analyzed to determine a similar dissociations, Kd = 5.5 and 6.4 μM. Thus, the conserved modifications appear to have little affect on the binding of paromomycin. Circular Dichroism (CD) experiments were also performed to corroborate the thermal transition data. Initial titrations of the human counterpart to Helix 69 with paromomycin indicate that the paromomycin does bind to the human rRNA (Kd = 18.4 μM), though not nearly as strongly as it binds with the prokaryotic (E.coli) RNA. While the E. coli Helix 44 and Helix 69 binding sites contain the A-platform which is required for paromomycin binding, the human Helix 69 site has a substituted G which may decrease the affinity of the aminoglycoside for that site. Current Research on this project includes the determination of thermodynamic parameters of the Helix 69-Paromomycin interaction using isothermal calorimetry (ITC). UV thermal titrations are also being performed to determine the interaction of Helix 69 with two other aminoglycosides, neomycin and tobramycin.

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