mRNA undergoes several essential modifications, the first of which is addition of a 7-methylguanosine cap. Because capping occurs soon after transcription initiation and is specific to RNA polymerase II transcripts, we postulated that the capping and transcription machinery are physically or functionally associated. Using a combination of biochemical and genetic techniques, we showed that capping enzyme is recruited to the initiation complex by interacting specifically with the phosphorylated C-terminal domain (CTD) of RNA polymerase II. We will extend these observations to map interaction domains of the CTD and capping enzyme, determine which kinase(s) are responsible, how CTD binding affects capping activities, and at which point capping enzyme is released from the transcription complex. Yeast capping enzyme consists of two proteins: a triphosphatase and a guanylyltransferase. In contrast, higher eukaryotic capping enzymes consist of a single protein with both activities. A genetic analysis of the yeast triphosphatase gene CET1 will be carried out, beginning with isolation of conditional alleles and site-directed mutagenesis. The mutants will be characterized to identify regions important for triphosphatase activity and for interaction with the guanylyltransferase subunit. We will also analyze a second yeast gene that encodes a protein similar in sequence to the capping enzyme triphosphatase. To extend our studies of capping to higher eukaryotes, we cloned and analyzed the C. elegans capping enzyme gene. This protein has one region homologous to yeast guanylyltransferases and a second with similarity to the protein tyrosine phosphatase (PTP) family. This domain has mRNA triphosphatase activity and we will determine whether a PTP-like mechanism is used. We will also characterize the mouse capping enzyme, which can function in yeast. We have identified several PTP-like proteins with extensive similarity to the capping enzyme triphosphatase and will test them for activity on RNA. mRNA capping is a fundamental process in gene expression. Although many aspects are conserved over evolution, there appear to major differences between yeast and higher eukaryotic capping enzymes that could be exploited for antifungal drug targeting.
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