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Date of Award


Document Type

Masters Thesis

Degree Name

Master of Science (MS)



First Advisor

Spiegel, P. Clint

Second Advisor

Anthony-Cahill, Spencer J.

Third Advisor

Prody, Gerry


Throughout all domains of life, each protein in a cell is synthesized by a remarkable biomolecular machine called the ribosome, in a process referred to as translation. This process is regulated by proteins called translation factors, several of which belong to the GTPase superfamily of enzymes which require the binding and subsequent hydrolysis of guanosine 5'-triphosphate (GTP) to execute their function. In contrast to the regulatory role of translation factors, protein biosynthesis is inhibited by several naturally occurring antibiotics. While our understanding of translation has been revolutionized by the recent elucidation of atomic-resolution x-ray crystal structures of the ribosome trapped in various intermediate conformations, several crucial aspects of protein biosynthesis remain poorly understood, such as the identity of the molecular component of the ribosome which stimulates the activation of the translational GTPases, as well as the mechanism by which several antibiotics inhibit translation. The major aims of this work are twofold. First, investigations directed towards the elucidation of the ribosomal element responsible for GTPase activation are described. It is demonstrated that the depletion of a specific protein from the ribosome which is part of the GTPase binding site, L12, results in significant attenuation of ribosome-dependent GTP hydrolysis activity by translational GTPases IF2, EF-G, LepA, and RF3, and this lost activity is fully restored by preincubating L12-depleted ribosomes with purified L12 protein. However, L12 alone does not stimulate GTP hydrolysis by these GTPases, in contrast to a previous report (Savelsbergh et al., 2000). In fact, it is shown that none of the isolated rRNA or protein components which comprise the ribosomal GTPase binding region stimulate GTP hydrolysis by the translational GTPases, implying that the peripheral ribosomal architecture is needed for correct positioning of the GTPase-activating element of the ribosome. A second major goal of this work was to investigate the inhibitory mechanism of the antibiotic thiostrepton, which is known to interfere with the function of elongation factor EF-G, and has been recently shown to inhibit the growth of the malarial parasite Plasmodium falciparum. Many lines of evidence reported herein contradict the current predominantly accepted model of thiostrepton action. It is shown that thiostrepton strongly inhibits ribosome-dependent GTP hydrolysis by EF-G and a closely related GTPase LepA, and this is explained by results which indicate that thiostrepton obstructs the binding of these factors to the ribosome. Interestingly, an engineered mutant of EF-G lacking domains IV and V is insensitive to thiostrepton, which is in agreement with recent structural evidence which suggests that thiostrepton interferes with the interaction between domain V of EF-G and the ribosome.




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