9316644 Butler We have identified and characterized an isolate of the yeast Saccharomyces cerevisiae having a conditional defect in the pathway for the production of mature mRNA. The recessive, lethal mutation (pap1-1) lies in the gene encoding poly(A) polymerase and results in thermolabile mRNA polyadenylation activity in vitro and in vivo. Shift of pap1-1 cells to the restrictive growth temperature results in a rapid loss of polyadenylated mRNA and the eventual death of the cell. We intend to exploit this mutation and the clone of the normal copy of the gene we obtained to pursue several lines of investigation designed to gain a better understanding of the function and mechanism of polyadenylation. This novel mutation and the strength of the genetic and biochemical techniques offered by this system put us in a unique position to bring new insights to this area of molecular biology. First, we will continue to characterize the pap1-1 mutant to determine why lack of polyadenylation kills the cells, thereby providing insight into the role of poly(A) tails in the cell. Second, we will search for and characterize suppressor mutations restoring the ability of pap1-1 cells to grow at the non-permissive temperature with the aim of identifying and understanding genes whose products interact with poly(A) polymerase, or play a role in mRNA metabolism. %%% This research has as its central aim the understanding of how cells modify the chemical structure of genetic messages, called messenger ribonucleic acids (mRNAs), and how these modifications affect the function of these molecules. Specifically, we are using to use baker's yeast as a model organism to study how the cell adds a series of nucleosides called adenosines to the end of mRNAs and to what degree this modification affects the ability of these messengers to translate their genetic information into proteins. During the period of our previous NSF award we isolated and characterized a yeast mutation that allows us to control, in living cells, the activity of the enzyme required to carry out this modification. Our approaches therefore feature the use of this mutation under conditions that allow us to study the synthesis and function of the modified mRNAs. Baker's yeast has proved to be an invaluable tool to study basic processes in cells because it is easy to grow and is amenable to biochemical and genetic analyses. Importantly, the basic molecular details of how cells grow and differentiate appear to be remarkably similar in yeast and humans, thus implying that basic biochemical research in yeast will shed light on the nature of basic molecular biological processes in many organisms as well. ***
