The bacterial ribosome is a target for inhibition by numerous antibiotics. Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E.coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio labeled antibiotics both in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained 16S and 17S rRNA. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug in vivo. In addition, cell free reconstitution assays generated a 21S particle during incubation with either aminoglycoside. Precursor formation was inhibited with increasing drug concentration. This work examines features of a novel antibiotic target for aminoglycoside and will provide information that is needed for the design of more effective antimicrobial agents.

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<p>Our understanding of the mechanism of ribosome assembly in bacteria is still in its infancy. Work from our laboratory and others have recently established that some protein assembly factors assist the assembly process at its late stages, mediating the correct folding of the functional core of the 30S and 50S subunits. The GTPase YjeQ is an assembly factor that displaces the upper domain of h44 of the mature 30S subunit upon binding, inducing a distortion in the decoding center. We hypothesized that the displacement of h44 is caused by the zinc-finger domain of YjeQ and mediates the release of RbfA, another assembly factor involved in 30S subunit maturation. To understand how the zinc-finger domain of YjeQ implements the functional interplay with RbfA, we constructed several deletion mutants of the domain. We found that the zinc-finger domain of YjeQ was required to bind the 30S subunit, but not the C-terminal extension (CTE) of the domain. The CTE was necessary for stimulation of GTPase activity upon binding to the 30S subunit and removal of bound RbfA from the 30S subunit. The data presented here suggests that the zinc-finger domain is essential for YjeQ to bind the 30S subunit and to implement the functional interplay with RbfA. Ongoing structural studies of the complex formed by the YjeQ CTE variant and the 30S subunit will provide a three dimensional view of the conformational changes that occur to implement the functional interplay between YjeQ and RbfA at the late stages of 30S subunit assembly.</p> / Master of Science (MSc)

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The overall objective of this dissertation research was to understand the mechanisms by which serum insulin-like growth factor-I (IGF-I) is regulated by nutritional intake in cattle. Two studies were conducted to achieve this objective. In the first study, effects of feeding levels on basal and growth hormone (GH)-stimulated serum concentrations of IGF-I, IGF binding protein-3 (IGFBP-3) and acid-labile subunit (ALS), and their mRNA expression in the liver were determined in beef cows. It was found that increased nutritional intake did not alter basal concentrations of serum IGF-I, IGFBP-3 or ALS, or their mRNA expression in the liver. However, under increased nutritional intake, GH administration stimulated a greater increase in serum IGF-I concentration, and this greater increase was not due to reduced degradation of IGF-I in serum. Increased nutritional intake did not enhance GH-stimulated IGF-I mRNA expression in the liver, but it increased the amount of IGF-I mRNA associated with polysomes, suggesting that liver translation of IGF-I mRNA is enhanced under increased nutritional intake. Under increased nutritional intake, GH also stimulated greater increases in serum IGFBP-3 and ALS concentrations, but these greater increases were not due to greater expression or translation of their mRNAs in the liver. Taken together, these results suggest that translation of GH-stimulated IGF-I mRNA in the liver is enhanced under increased nutritional intake and this enhancement may be partially responsible for the greater GH-stimulated increase in serum IGF-I concentration. These results also suggest that the greater GH-stimulated increases in serum IGFBP-3 and ALS may be secondary to the greater increase in serum IGF-I because increased IGF-I may increase the formation of IGF-I/IGFBP-3/ALS complexes, thereby increasing the retention of IGFBP-3 and ALS in the blood. In the second study, the effects of food deprivation on serum IGF-I concentration in steers and the underlying mechanism were determined. It was found that food deprivation decreased serum IGF-I concentration and that this decrease was not due to increased IGF-I degradation in serum. Food deprivation decreased liver IGF-I mRNA expression, and this decrease was associated with decreased expression of GH receptor (GHR) mRNA and protein in the liver. Food deprivation was also associated with increased mRNA expression of two inhibitors of the GHR signaling pathway, suppressor of cytokine signaling-2 (SOCS2) and cytokine-inducible SH2 protein (CIS). These results suggest that decreased IGF-I gene expression in the liver may be at least partially responsible for the decrease in circulating IGF-I concentration during food deprivation, and that the former decrease may be due to increased expression of SOCS2 and CIS, and decreased expression of GHR in the liver. Overall, this dissertation research indicates that multiple mechanisms are involved in nutritional regulation of circulating IGF-I concentration in cattle. / Ph. D.