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

酵母mRNA capping酶由Ceg1和Cet1组成，分别负责mRNA guanyyltransferase （GTase）和RNA 5'-triphosphatase （TPase）的活性。为了研究Cet1的结构和功能，我们分离并鉴定了温度敏感（ts）突变体。为了在单拷贝质粒上分离cet1^<ts>突变体，将YCpW-CET1用羟胺处理后转化为SK1， SK1是一种染色体cet1Δ：： LEU2干扰物，携带外体多拷贝质粒，YEp-CET1含有野生型cet1。摘取转化子，在25℃的FOA（5-氟乙酸）板上培养，进行质粒洗牌，表达突变的Cet1。我们分离了7个温度敏感的CET1突变体（CET1 ^<ts>-1至CET1 ^<ts>-7）。根据缺失突变分析，所有这些突变都位于TPase活性必需区（265-549）和Cet1-Ceg1相互作用必需区（205-265）。我们在大肠杆菌中表达所有cet1^<ts>突变蛋白作为GST融合，并检测这些RNA 5'-三磷酸酶活性。3个ts突变体G257D （cet1^<ts>-1）、S419L （cet1^<ts>-2）、T396I/T400I （cet1^<ts>-3）具有足够的TPase活性。与这些突变体相反，R532K突变体使TPase活性大大降低，而ts突变体则没有。这些结果表明，TPase的还原与温度敏感性之间存在差异。一个有趣的突变（R242K）产生了这个突变体。但R242K/A257N或R242K/E200K双突变看起来生长正常。结果表明，氨基酸A257、R242和E200与Cet1-Ceg1相互作用存在协同作用。

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.