Decoding of genomic information, so basic to the biology of protein synthesis in all cells and seemingly so simple, now is complicated by the finding that it requires a diverse set of chemical additions to the RNA molecules that are involved, transfer RNAs (tRNAs). tRNAs are responsible for translating the genetic information for a protein into the amino acid sequence of that protein. Many tRNAs devoid of these chemical modifications, particularly at two different positions (the anticodon wobble position-34 and/or the purine nucleoside-37), will not recognize the amino acid code incorporated into messenger RNA. The chemical modifications at these two positions in the tRNAs rescue tRNAs' ability to read the amino acid codes, codons. The tRNAs for amino acids that have two codons have to be restricted in their reading so that they do not read the codons of other amino acids. The tRNAs for amino acids that have four codons have to have the ability to read the four codons. Significantly, these two sets of tRNAs are differently modified. Thus, the proposal's long-term objectives are to a) Establish general principles for tRNA's modification-dependent decoding of codons; b) Educate and excite young scientists to the chemistries of RNA by integrating education with science; c) educate the scientific community as to the importance of modifications tRNA function; and d) apply an understanding of modification structure/function relationships to designing RNA tools in molecular and cellular biology. The research is focused on the physical chemical mechanism(s) by which modifications may control tRNA's recognition of two versus four codons. Do distinctly different modifications contribute to tRNA's ability to decode two versus four-fold codons by restricting codon recognition in the former and expanding wobble in the latter? Does codon recognition require that the protein synthesizing machinery, the ribosome, induce a correct fit architecture to tRNA? Do modifications pre-form the tRNA structure for protein synthesis? To answer the first question modifications that restrict tRNAs binding to two codons will be compared to the binding of differently modified tRNAs to four codons on the ribosome. To answer the second and third questions, the affects of the various modifications tRNA stability, and structure in solution, and structure on the ribosome will be related to their ability to decode. Solutions structures from nuclear magnetic resonance spectroscopy will be compared to each other and to the structures bound to codon on the ribosome. These experiments will determine whether modifications pre-structure the tRNA prior to codon binding, and whether the ribosome induces architecture for codon binding.
An understanding of the modification-dependent mechanism of tRNA's reading of the Genetic Code is fundamental to biochemistry, cell biology and to the education of young scientists. A new structural-based model of wobble decoding on the ribosome with modification-dependent, novel base pairing geometries will be generated. The model will include either a modification-dependent pre-formed or ribosome induced-fit anticodon architecture.
The broader impact of the project is focused on the educational objective to instill scientific curiosity in young minds, and research experience in young hands by exciting their involvement in answering questions about RNA biology. By integrating research with education undergraduate and graduate students at North Carolina State University and North Carolina Agricultural and Technical State University, a historically black college, will learn scientific methodology from peers and mentors. To the benefit of society underrepresented minorities and women will be mentored in physical biochemistry; they represent a significant proportion (12%) of the Department's present majors. NCSU's research infrastructure will be enhanced by purchase of a microcalorimeter for the physiochemical study of RNA. The collective housing of a number of instruments will establish a shared educational infrastructure and creates a supportive environment for interactions and mentoring among Triangle Universities dedicated to RNA research.
