The formation of mRNA 3' ends involves precise cleavage of precursor followed by addition of adenylate residues to the new end of the RNA. The primary goal of this research is a thorough molecular analysis of polyadenylation in the yeast S. cerevisiae, using a combination of genetic and biochemical approaches. An understanding of the mechanism of polyadenylation will provide the basis from which to ask questions about how the process is regulated as the physiological state of the cell changes, how this regulation would affect mRNA levels globally or specifically, and how polyadenylation interacts with other processes involved in mRNA synthesis. The first specific aim is to biochemically characterize factors involved in yeast rnRNA polyadenylation and to understand at a molecular level how these factors interact with each other and with the precursor RNA. These factors will be purified to homogeneity, using in vitro assays for each step in the processing reaction Peptide sequence or monoclonal antibodies derived from purified components will be used to clone the genes encoding these factors. The second specific aim is to use genetic analysis to study trans-acting factors necessary for polyadenylation. The following screen will be used to detect mutations in processing factors. Yeast strains which are temperature sensitive for growth will be screened for conditional phenotypes in three types of assays: i) production of blue colonies due to transcriptional read-through into beta-galactosidase coding sequences, 2) Northern analysis of RNAs made in vivo from a construct designed to give a discrete poly(A)- read-through product whose end is specified by snRNA termination signals, and 3) examination of extracts made from the best candidates to identify ones which are reproducibly defective for processing in vitro. The genes can be cloned by complementation of the conditional defect with a wild-type yeast genomic library. The genetic analysis will aid the biochemical characterization in several important ways. If one of the factors cannot be cloned by protein sequence or antibody screening, it may be possible to identify it with a genetic screen. Gene disruption will indicate if the proteins identified biochemically are essential for viability in yeast. Finally, genetic strategies such as suppressor analysis, identification of pairs of mutant genes which exhibit synthetic lethality, and directed mutagenesis of the genes, can be used to determine how these factors interact in vivo, and to assign functions to different domains of the proteins. The final specific aim is to further define the signals which specify polyadenylation in yeast. Random PcR and chemical mutagenesis will be used to determine what sequences in addition to the (UA)6 repeat are essential for GAL7 polyadenylation. Debilitating mutations will be detected in vivo using a beta-galactosidase reporter construct, and then tested in vitro for their effects on cleavage and/or poly(A) addition.
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