Recoding of the genetic code is subject to redirection and redefinition at specific sites in a small number of mRNAs for a wide range of organisms. Redefinition of termination codons can be used to produce larger functional proteins, or alternatively, to insert specialized amino acids (i.e. selenocysteine) into specific points of a protein for altered physical properties. Recoding is under control of information embedded within the mRNA, which is available as specific codons and structured regions in the mRNA (stimulators). A structural understanding of these mRNA stimulators will give insight to the overall mechanisms of redefinition events and a general understanding for translational recoding. A structural investigation of two mRNA stimulatory elements is proposed. (1) The stimulatory element required for selenocysteine insertion in eukaryotes is located in the 3'-UTR and is composed of a stem loop structure with an internal loop. The conserved core region and run of adenosines in the apical loop will be investigated by NMR in terms of structural properties these conserved regions possess. This will clarify discrepancies of two proposed secondary models, and will highlight the importance of loop I in aligning the two helical regions about the core region. (2) The pseudoknot stimulatory element shown to be required for UAG redefinition in Maloney Murine Leukemia Virus (MuLV) will be biophysically probed to tune up the current secondary model produced by genetic approaches, and to interpret highly conserved regions in the proposed loopII and spacer regions. Experiments will be conducted with the presence of a additional element upstream of the UAG termination codon. Competition for the formation of the pseudoknot will be monitored, as well as the efficiency of redefinition. Cellular components that interact with either/both of these elements will be sought through the life cycle of this retrovirus. This information will enable us to produce small model oligonucleotides for high-resolution studies of this pseudoknot stimulator.
