Sructural and function analysis of yeast mRNA capping enzyme in transcription reaction.

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The yeast mRNA capping enzyme is composed of Ceg1 and Cet1, responsible for the activities of mRNA guanylyltransferase (GTase) and RNA 5'-triphosphatase (TPase), respectively. To investigate structure and function of Cet1 , we had isolated and characterized temperature sensituve (ts) mutants. For isolation of cet1^<ts> mutants on single-copy plasmid, YCpW-CET1 was treated with hydroxylamine and transformed into SK1 which is a chromosomal cet1Δ : : LEU2 disruptant carrying with episomal malti-copy plasmid, YEp-CET1 containing wild type CET1. Transformants were picked and grown on FOA (5-fluoroorotic acid) plates at 25℃ to carry out plasmid shuffling and to express mutated Cet1. We isolated 7 temperature-sensitive mutants of CET1 (cet1^<ts>-1 to cet1^<ts>-7). All these mutations located in the essential for TPase activity (265-549) and Cet1-Ceg1 interaction (205-265) regions based on deletion mutation analysis. We expressed all cet1^<ts> mutant proteins as GST fusion in E.coli and assayed these RNA 5'-triphosphatase activity. Three ts mutants, G257D (cet1^<ts>-1), S419L (cet1^<ts>-2), T396I/T400I (cet1^<ts>-3), had enough TPase activity. Contrary to these mutants, R532K mutation made greately reduced TPase activity but not ts mutant. These result indicated that it is different between reduction of TPase and temperature sensitivity. One interesting mutation (R242K) made ts mutant. but R242K/A257N or R242K/E200K double mutation looked growth normal. This data suggested that three amino acids (A257, R242 and E200) were working with Cet1-Ceg1 interaction cooperatively.