Ho C K, Sriskanda V, McCracken S, Bentley D, Schwer B, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
J Biol Chem. 1998 Apr 17;273(16):9577-85. doi: 10.1074/jbc.273.16.9577.
We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities. The principal conclusions are as follows: (i) the mammalian capping enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211-597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg1; (iii) the guanylyltransferase domain per se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1-210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II. These findings contribute to an emerging model of mRNA biogenesis wherein RNA processing enzymes are targeted to nascent polymerase II transcripts through contacts with the CTD. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals.
我们对小鼠mRNA加帽酶（Mce1）进行了生化和遗传学分析，Mce1是一种具有RNA三磷酸酶和RNA鸟苷酸转移酶活性的双功能597个氨基酸的蛋白质。主要结论如下：（i）哺乳动物加帽酶由自主且不重叠的功能结构域组成；（ii）鸟苷酸转移酶结构域Mce1(211 - 597)在体外具有催化活性，在酵母体内可替代内源性鸟苷酸转移酶Ceg1发挥功能；（iii）鸟苷酸转移酶结构域本身与磷酸化的RNA聚合酶II羧基末端结构域（CTD）结合，而三磷酸酶结构域Mce1(1 - 210)不与CTD结合；（iv）小鼠三磷酸酶活性位点半胱氨酸的突变在酵母中引发强烈的生长抑制表型，这可能是通过将前体mRNA末端隔离在无生产性的复合物中或通过阻止内源性酵母三磷酸酶接近RNA聚合酶II来实现的。这些发现有助于形成一种新出现的mRNA生物合成模型，其中RNA加工酶通过与CTD的接触被靶向到新生的聚合酶II转录本上。鸟苷酸转移酶与CTD之间的磷酸化依赖性相互作用从酵母到哺乳动物都是保守的。
