The fidelity of the protein synthetic apparatus is unquestionably of extreme importance in cell function, regulation and replication. Nonetheless errors apparently occur at frequencies several orders of magnitude above those in DNA synthesis. These errors may be involved in progressive diseases such as aging and may also play an important role in natural protein variability. Some viral messages must be """"""""mistranslated"""""""" in order for appropriate proteins to be synthesized.
The aim of the research proposed in this project is to do detailed analysis on the type and frequency of in vivo mistranslational events. Several model proteins, whose genes are carried on bacterial plasmids, will be studied: MS2 coat protein, ornithine transcarbamylase and yeast ribosomal protein L3. These proteins were chosen on the basis of codon usage in their message. Most studies will be done in Escherichia coli but some will also be carried out in the yeast Saccharomyces cerevisiae. The assay to be employed involves amino acid starvation to increase misreading of codons cognate for the deprived amino acid, separation of radiolabeled protein by polyacrylamide gel electrophoresis, electroelution and radiochemical microsequencing. Mistranslation will also be examined in cells experiencing """"""""heat shock."""""""" The internal concentration of tRNA will also be changed by using a tRNA gene which has been placed under the control of a synthetic lac promoter. The influence of codon context will be studied by altering the sequence of the gene(s) by site-directed in vitro mutagenesis. Neighboring bases and codons will be altered as well as potential regions of secondary structure.
