A study of site-specific modifications in ribosomal RNA (rRNA) is proposed in which a combination of synthetic, biophysical, and biological approaches will be taken. The long-term goal of the work is to understand the significance of modified nucleotides in rRNA and their roles in regulation of ribosome function, namely translation. The design of novel rRNA-targeting drugs as potential antibacterials will ultimately rely on a fundamental understanding of rRNA structure- function relationships. The short-term goals of the proposed project are to obtain detailed information regarding the roles of pseudouridine and its analogues in regulating ribosome conformation changes and dynamics as they relate to protein synthesis. Modified RNAs representing the exposed hairpin in domain IV of the bacterial large subunit rRNAs (helix 69) will be synthesized and characterized by various biophysical techniques. Preliminary studies revealed sequence and structural differences between human and bacterial H69 RNAs, and literature reports indicate that H69 is essential for normal ribosome function, making this region of the ribosome an ideal drug-targeting site.
The specific aims of the research plan are to: 1) characterize the conformational switching mechanism in H69 in humans and bacteria through a variety of biophysical studies (NMR, circular dichroism, fluorescence spectroscopy), 2) use chemical probing and biological assays on full-length ribosomal RNAs and ribosomes to reveal whether structural changes observed in model systems are relevant in natural systems, and 3) identify and characterize ligands that have specificity for bacterial helix 69 over the human variant, and can selectively inhibit bacterial ribosome function. Together these aims combine the strengths of multiple areas of research (organic synthesis, RNA modification, RNA biophysics, and chemical probing) t address key issues that will impact human health and defense against resistant bacteria.
The protein synthesis machinery, the ribosome, is essential to all living organisms, and catalyzes key steps in translating genetic information stored in the form of a nucleic acid to a functionally important protein. This machine depends on precise molecular interactions in order to maintain fidelity and carry out its function essentially without errors. The pseudouridine modification, which is abundant in ribosomal RNA, plays a key role in maintaining such fidelity. One region containing conserved pseudouridine modifications, helix 69, is essential for normal cell growth. The exact roles of pseudouridine in regulating ribosome function are currently unknown.
The aims of this proposal are to elucidate the molecular and physical basis for helix 69 dynamics and function, and to determine key differences between human and bacterial ribosome structure and function. This information will be used to design and select for ligands that selectively target bacterial ribosomes, and could lead to the development of potential therapeutics for drug-resistant and/or pathogenic bacteria, thus having an impact on human health.
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