Tsukamoto T, Shibagaki Y, Imajoh-Ohmi S, Murakoshi T, Suzuki M, Nakamura A, Gotoh H, Mizumoto K
Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
Biochem Biophys Res Commun. 1997 Oct 9;239(1):116-22. doi: 10.1006/bbrc.1997.7439.
The yeast Saccharomyces cerevisiae mRNA capping enzyme is composed of two subunits of alpha (52 kDa, mRNA guanylyltransferase) and beta (80 kDa, RNA 5'-triphosphatase). We have isolated the alpha subunit gene (CEG1) by immunological screening. In this report, with the aid of partial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designated CET1, encoding the S. cerevisiae capping enzyme beta subunit. Amino acid sequence analysis revealed that the gene encodes for 549 amino acids with a calculated M(r) of 61,800 which is unexpectedly smaller than the size estimated by SDS-PAGE. Gene disruption experiment showed that CET1 is essential for yeast cell growth. The purified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphosphatase activity which specifically removed the gamma-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene. Interaction between the Cet1 and the Ceg1 was also studied by the West-Western procedure using recombinant Ceg1-[32P]GMP as probe.
酵母酿酒酵母的mRNA加帽酶由α（52 kDa，mRNA鸟苷酸转移酶）和β（80 kDa，RNA 5'-三磷酸酶）两个亚基组成。我们通过免疫筛选分离出了α亚基基因（CEG1）。在本报告中，借助纯化的酵母加帽酶的部分氨基酸序列，我们分离出了编码酿酒酵母加帽酶β亚基的基因，命名为CET1。氨基酸序列分析表明，该基因编码549个氨基酸，计算所得的分子量（M(r)）为61,800，这比SDS-PAGE估计的大小意外地小。基因破坏实验表明，CET1对酵母细胞生长至关重要。纯化的重组CET1基因产物Cet1表现出RNA 5'-三磷酸酶活性，该活性可特异性地从三磷酸末端的RNA底物上去除γ-磷酸，但不能从核苷三磷酸上去除，从而证实了该基因的身份。还使用重组Ceg1-[32P]GMP作为探针，通过West-Western方法研究了Cet1与Ceg1之间的相互作用。
