The fidelity of the protein synthetic apparatus is unquestionably of extreme importantance in cell function, regulation, and replication. Many of the effects of the production of faulty protein can be readily imagined to have serious consequences to the cell, and could be involved in a large number of progressive diseases.
The aim of the research proposed in this project is to continue the detailed analysis of the in vivo, high-level mistranslation which occurs when bacterial or mammalian cells are starved for certain amino acids. Mistranslated protein species can readily be identified and quantitated on polyacrylamide gels. Since this phenomenon seems identical in both bacterial and mammalian systems, we shall use Escherichia coli and E. coli infected with the small RNA virus MS2 as model systems. We have demonstrated that most of the mistranslation we observe is the result of third position misreading of the genetic code. By using peptide and sequencing analysis of mistranslated protein, we will quantitatively assess the tendency of specific codons or codons in specific contexts to be misread. Construction of a functional double stranded DNA copy of the RNA phage coat protein gene will allow us to test the influence of message structure on mistranslation. Other errors (frameshift, nonsense) and other error producing conditions will also be examined. We shall catalog the frequency of errors in several proteins and also the conditions (specific mutations or antibiotics) which influence these frequencies. Specific aminoacyl tRNA synthetases and tRNA's will be manipulated genetically to determine what effects these have on the nature and frequency of errors. The activity of certain mistranslated proteins will be also assessed.
