Structural studies on the interaction of nucleosomes with metal-based anticancer compounds and the high mobility group A2 protein and X-ray crystallographic investigation of bacterial ribosome translocation
Date of Issue2016
School of Biological Sciences
The mystery of life lies in its ability to transform genetic information into biological traits and passage this information through generations of organisms as heritable material. The “central dogma” of molecular biology proposes that cells utilize intricate molecular machinery to express genetic information as a phenotype by converting archival molecules into functional molecules. In the “central dogma” scheme this genetic information, initially stored in nucleic acid polymers as DNA, is transcribed into the alternate nucleic acid RNA before it is finally translated into protein products. This thesis will provide structural insights into this complex machinery by exploring three of its operational components. First, the nucleosome, the effective unit of stored genomic information, is probed as a target for metal-based anticancer agents. Second, the interaction of high mobility group AT-hook 2 with the nucleosome and its influence on nucleosomal function is described. And finally, the behavior of ribosomal translation machinery under varying circumstances during translocation will be clarified. Unlike prokaryotes, eukaryotes pack their genomic DNA into chromatin. The basic repeating unit of chromatin, the nucleosome, plays essential roles in storing genomic information and modulating transcriptional activities. Conformational features and epigenetic variations of the nucleosome, and its subjection to strict cellular regulation, make it an attractive target for anticancer drug development. Ruthenium anticancer compounds have been found to yield high selectivity and low toxicity. Crystallographic analysis of the ruthenium-arene agent, [(η6-THA)Ru(II)(ethylene-diamine)Cl][PF6] (THA=5,8,9,10-tetrahydroanthracene), identified a novel intercalation mode in the nucleosomal DNA. Ruthenium(II)-arene-1,3,5-triaza-7-phosphaadamantane (RAPTA) compounds have been found to specifically target histone protein sites in chromatin. Our studies of several new dinuclear RAPTA-based agents reveal further links between chemical structure and activity. Furthermore, the synergistic potential of metalloagent combinations were examined in crystal structures of nucleosomes adducted with a binuclear platinum- and ruthenium-based bifunctional ligand, and with a RAPTA compound plus gold-based agent bimolecular combination. HMGA2, a chromatin architectural factor, is involved in numerous nuclear pathways. HMGA2’s transcription modulating activity arises from its ability to alter chromatin conformation. However, despite its mechanical impact on chromatin, there is no structural information available regarding its interaction with the nucleosome. As HMGA2 favors AT-rich regions of nucleosomal DNA, we designed and prepared different nucleosomal constructs with poly-A linker DNA and verified the protein’s affinity for these regions. Additionally, numerous crystallization trials were performed under a variety of conditions. These trials also yielded the first report for a crystal structure of a nucleosome, which is a 167 bp construct containing eleven base pairs of linker DNA at each terminus. The results of this project provide a better understanding of HMGA2’s functional activities, nucleosome structure and shed light on new crystallization approaches for structural characterization of nucleosomal assemblies. The ribosome is the fundamental machinery for protein translation in the cell. During the process of translation elongation, genomic information is converted from mRNA into polypeptide chains. Elongation factor G (EF-G) is a GTPase involved in the translocation of A- and P-site tRNA to the P- and E-sites on the ribosome respectively, while a newly defined elongation factor, Leader peptidase A (LepA or EF4), is able to trigger back-translocation of the ribosome upon activation of its own ribosome-dependent GTPase activity. The translocation and back-translocation functions of these two factors are well characterized by biochemical studies, however, detailed atomic level information of these mechanisms is still lacking. Our efforts focused on exploring crystal structures of EF-G or LepA bound bacterial ribosome complexes in pre-GTP hydrolysis states. Additionally, we also made progress on a ribosome-stalling-rescue factor YaeJ, which releases abortively synthesized peptide chains to reduce their toxic accumulation. The binding of these factors and their occupancy within ribosome complexes were confirmed and crystals were obtained. These studies lend novel insights into how ribosomes account for fluctuating conditions during translation.