Structure and Function of Eukaryotic Ribosomes

Brown University, Providence RI

Investigators

Abstract

Intellectual Merit: The objective of this research is to identify conserved nucleotide elements (CNEs) in ribosomal RNA (rRNA) and elucidate their functions in the ribosome. The CNE regions must be crucial for ribosome biogenesis, structure and/or function as they have been maintained throughout evolutionary time. Presumably, mutations in these regions would have been lethal to the organism and hence not perpetuated. This research project builds upon previous NSF-funded research in which a Complete Organismal rRNA Database (CORD) was developed and bioinformatic methods were designed to identify CNEs in the large ribosomal subunit rRNA. These methods were applied to eukaryotic 25-28S rRNAs and 42 CNEs were thus identified. Of these 42 CNEs, five are universally conserved in all Domains of life (eukaryotes, bacteria and archaea), and nine other CNEs are specific to eukaryotes with their sequences being degenerate in bacteria. This analysis will be expanded to identify CNEs in bacterial 23S rRNA and to discern which bacterial CNEs are universally conserved in all domains of life and which are specific to bacteria. Mutations will be made in each of the nine eukaryotic-specific CNEs to analyze their function in rRNA processing, ribosome export, ribosome function in protein synthesis and coordination with the cell cycle. Furthermore, an MS2-based pulldown strategy will be developed for isolation of ribosomes containing mutations in the rRNA. Development of this system will provide the foundation for future proteomic and structural studies on ribosome biogenesis. The results of the research described here will be of great value to the scientific community. rRNA is used as a yardstick for phylogenetic comparisons, so the derivation of CORD will be useful to evolutionary biologists. Moreover, the results will be very valuable to molecular biologists in their study of ribosomes. The next frontiers of ribosome research are (i) a study of eukaryotic ribosomes, (ii) a full description and understanding of ribosome biogenesis and (iii) elucidation of conformational changes that occur in ribosomes during elongation in protein synthesis. The data from the research described here will highlight the regions in rRNA of great functional importance for these processes, thus helping to focus studies on structure and function by the scientific community. Broader Impacts: The research described here will be integrated with education, serving to train undergraduates through a postdoctoral associate who will help with this research. Graduate education and postdoctoral training are topics of great interest to the Principal Investigator who has played leadership roles at the national level in this area. The research described here will help to broaden participation in science by women and minorities, with students from these groups working on the research. Furthering the opportunities in science for women and minorities is a topic in which the Principal Investigator has been actively involved at Brown University and at the national level. New methods and tools will be developed by the research described here, thus enhancing the infrastructure for scientific research. The results will be disseminated to the scientific community through publications and talks. The Principal Investigator has a track record of bringing the benefits of biological research to the attention of the lay public and to Congress.