Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism.
Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism.
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DOI：
10.1073/pnas.95.21.12226
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发表时间：
1998-10
影响因子：
11.1
通讯作者：
Y. Wen;Z. Yue;A. Shatkin
中科院分区：
文献类型：
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作者：
Y. Wen;Z. Yue;A. Shatkin
哺乳动物加帽酶是具有RNA 5 '-三磷酸酶和鸟苷酰转移酶活性的双功能蛋白质。N-末端237-aa三磷酸酶结构域包含（I/V）HCXXGXXR（S/T）G，其序列对应于蛋白酪氨酸磷酸酶（PTP）中的保守活性位点基序。对小鼠RNA 5'-三磷酸酶的点突变体的分析鉴定了基序Cys和Arg残基以及活性所需的上游Asp。与PTP类似，该酶被碘乙酸盐和VO43-抑制，并且不依赖于Mg 2+，这为PTP样机制从RNA 5 '末端去除磷酸盐提供了额外的证据。与三磷酸酶结构域不同，全长597-aa小鼠加帽酶和C-末端鸟苷酰转移酶片段（残基211 - 597）结合聚（U），并且在转染细胞中为核。GTP增加RNA结合，鸟苷酸化缺陷，活性位点突变体不受影响。在形成酶-GMP加帽中间体（R315、R530、K533或N537）所需位置的Ala取代也消除了poly（U）结合，而在这些位点具有保守取代的蛋白质保留了结合活性，但不保留鸟苷酰转移酶活性。这些结果表明，哺乳动物加帽酶的鸟苷酰转移酶结构域指定核定位和RNA结合。加帽酶与新生转录物的结合可以与RNA聚合酶II结合协同作用以确保5 '帽形成。
Mammalian capping enzymes are bifunctional proteins with both RNA 5'-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I/V)HCXXGXXR(S/T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5'-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity. Like PTPs, this enzyme was inhibited by iodoacetate and VO43- and independent of Mg2+, providing additional evidence for phosphate removal from RNA 5' ends by a PTP-like mechanism. The full-length, 597-aa mouse capping enzyme and the C-terminal guanylyltransferase fragment (residues 211-597), unlike the triphosphatase domain, bound poly (U) and were nuclear in transfected cells. RNA binding was increased by GTP, and a guanylylation-defective, active-site mutant was not affected. Ala substitution at positions required for the formation of the enzyme-GMP capping intermediate (R315, R530, K533, or N537) also eliminated poly (U) binding, while proteins with conservative substitutions at these sites retained binding but not guanylyltransferase activity. These results demonstrate that the guanylyltransferase domain of mammalian capping enzyme specifies nuclear localization and RNA binding. Association of capping enzyme with nascent transcripts may act in synergy with RNA polymerase II binding to ensure 5' cap formation.