Eukaryotic messenger RNAs contain the 5' cap structure m7GpppN. Capping occurs by a series of three enzymatic reactions in which the 5' triphosphate terminus of a primary transcript is first cleaved to a diphosphate terminated RNA by RNA triphosphatase, then capped with GMP by RNA guanylyltranferase, and subsequently methylated at the N7 position of guanine by RNA (guanine-7) methyltransferase. The goal of this project is to understand the biochemistry of cap formation and the role of the cap in cellular RNA metabolism. This will be accomplished through biochemical and molecular genetic analysis of the enzymes involved in cap synthesis, using the budding yeast Saccharomyces cerevisiae as a model system. The yeast CEG1 gene encodes GTP:mRNA guanylytransferase, an essential 52kD protein that caps the RNA 5' end in a two-stage reaction involving a covalent enzyme-nucleotidyl intermediate. The capping intermediate, consisting of GMP linked to the epsilon-amino group of Lys-70 of the CEG1 protein, resembles the enzyme-AMP intermediate formed by DNA and RNA ligase.
One aim of the proposal is to map structural elements of the CEG1 protein that are essential for covalent catalysis and, in doing so, to address whether sequence conservation between capping enzyme and polynucleotide ligase is relevant to the common on catalytic mechanism. To better understand the mechanism and regulation of cap synthesis, the yeast genes encoding the triphosphate and methyltransferase components will be cloned by """"""""reverse genetic."""""""" This will entail purification of the triphosphatase and methyltransferase activities, peptides sequencing, and the design of oligonucleotide probes for isolation of the relevant genes. Efforts to define the role of the cap in RNA metabolism will take advantage of a collection of temperature-sensitive ceg1 mutations. Although it has been suggested that the cap structure may target mRNAs for processing, transport, and translation, as well as protect mRNA from degradation, there are few in vivo studies to substantiate these assumptions. By following the fate of specific transcripts that are synthesized in ceg1-ts cells after shift to the nonpermissive temperature, the consequences of the lack of RNA capping (or capping enzyme) on """"""""downstream"""""""" RNA transactions will be deduced. Specific parameters to be examined include pre-mRNA splicing, mRNA decay, and poly(A) addition. Proteins that interact functionally with CEG1 or impact on cap- dependent events will be identified by the isolation and characterization of extragenic suppressor of ceg1 mutations.
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