Charkaburtty The central theme of this proposed research is to investigate the molecular mechanism of the novel yeast elongation factor 3 (EF-3) uniquely required by yeast ribosomes for translation. A unique structural feature of EF-3 is the presence of the duplicated ATP-binding cassettes (ABC or NBS motifs) similar to that present in transporter proteins such as CFTR and multidrug resistant protein MDR. EF-3 is an ATPase. It's function in translation requires ATP hydrolysis. The intrinsic ATPase activity of EF-3 is stimulated two orders of magnitude by yeast ribosomes. In this project, the PI will investigate the function of the duplicated ATP-binding sequence motifs in translation by oligo-directed mutagenesis studies. The role of these motifs will be analyzed by directed mutation of the invariant lysine residue in motif-A, the aspartate in motif-B and the invariant glycine in motif-C. Each of these amino acid residues has been shown to coordinate with ATP. Na-orthovanadate will be used to selectively inactivate one of the two NBS motifs. The cooperative or independent function of the two NBS motifs will be analyzed by trapping the Mg-ADP-orthovanadate complex preferentially at one site. A battery of biochemical tests are available to analyze the effects of NBS mutation in EF-3 function. The second objective of this research will be to initiate x-ray crystallographic analysis of the ATP-binding domain of EF-3. The structural analysis will be initiated in collaboration with the macromolecular crystallography group at the University in Aarhus, Denmark. The PI will attempt to crystallize the full length EF-3 and the ATP-binding domain in the presence and absence of ATP analogs. The ATP-binding domain will be expressed in an E. coli expression system to obtain seleno-methionine substituted EF-3. This substitution allows rapid structure determination by the multiple anomalous dispersion method.
Deciphering the function of EF-3 at the molecular level can be expected to provide new insights into the biochemical mechanism of translation-elongation and the role of EF-3 in maintaining translational accuracy. YEF3 gene is essential for the growth of fungi. The indispensable role of EF-3 in fungi remains an enigma in the face of its apparent dispensability in all other non-fungal systems. Clearly, the question is what protein or RNA species replace(s) the function of EF-3 in higher eukaryotes. In order to determine whether there is an equivalent factor in other systems, it is important to know more about this protein. It will be interesting to learn why mammalian ribosomes do not require EF-3. The answer to this last question is of significant practical importance as EF-3 is a potential target for anti-fungal drugs. The availability of the x-ray crystal structure data of EF-3 and its ATP-binding domain is expected to further our understanding of its binding site on the ribosome. The information, in turn, should provide better understanding of the function of EF-3 in translation.
