Lehman K, Schwer B, Ho C K, Rouzankina I, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, NY, USA.
J Biol Chem. 1999 Aug 6;274(32):22668-78. doi: 10.1074/jbc.274.32.22668.
The 549-amino acid yeast RNA triphosphatase Cet1p catalyzes the first step in mRNA cap formation. Cet1p consists of three domains as follows: (i) a 230-amino acid N-terminal segment that is dispensable for catalysis in vitro and for Cet1p function in vivo; (ii) a protease-sensitive segment from residues 230 to 275 that is dispensable for catalysis but essential for Cet1p function in vivo; and (iii) a catalytic domain from residues 275 to 539. Sedimentation analysis indicates that purified Cet1(231-549)p is a homodimer. Cet1(231-549)p binds in vitro to the yeast RNA guanylyltransferase Ceg1p to form a 7.1 S complex that we surmise to be a trimer consisting of two molecules of Cet1(231-549)p and one molecule of Ceg1p. The more extensively truncated protein Cet1(276-549)p, which cannot support cell growth, sediments as a monomer and does not interact with Ceg1p. An intermediate deletion protein Cet1(246-549)p, which supports cell growth only when overexpressed, sediments principally as a discrete salt-stable 11.5 S homo-oligomeric complex. These data implicate the segment of Ceg1p from residues 230 to 275 in regulating self-association and in binding to Ceg1p. Genetic data support the existence of a Ceg1p-binding domain flanking the catalytic domain of Cet1p, to wit: (i) the ts growth phenotype of 2mu CET1(246-549) is suppressed by overexpression of Ceg1p; (ii) a ts alanine cluster mutation CET1(201-549)/K250A-W251A is suppressed by overexpression of Ceg1p; and (iii) 15 other cet-ts alleles with missense changes mapping elsewhere in the protein are not suppressed by Ceg1p overexpression. Finally, we show that the in vivo function of Cet1(275-549)p is completely restored by fusion to the guanylyltransferase domain of the mouse capping enzyme. We hypothesize that the need for Ceg1p binding by yeast RNA triphosphatase can by bypassed when the triphosphatase catalytic domain is delivered to the RNA polymerase II elongation complex by linkage in cis to the mammalian guanylyltransferase.
由549个氨基酸组成的酵母RNA三磷酸酶Cet1p催化mRNA帽形成的第一步。Cet1p由三个结构域组成：(i) 一个230个氨基酸的N端片段，该片段在体外催化以及Cet1p在体内发挥功能时都是可有可无的；(ii) 一个对蛋白酶敏感的片段，从第230位氨基酸到第275位氨基酸，该片段对催化作用可有可无，但对Cet1p在体内发挥功能至关重要；(iii) 一个催化结构域，从第275位氨基酸到第539位氨基酸。沉降分析表明，纯化的Cet1(231 - 549)p是一种同型二聚体。Cet1(231 - 549)p在体外与酵母RNA鸟苷酸转移酶Ceg1p结合，形成一个7.1 S的复合物，我们推测该复合物是由两个Cet1(231 - 549)p分子和一个Ceg1p分子组成的三聚体。截短程度更大的蛋白质Cet1(276 - 549)p不能支持细胞生长，以单体形式沉降，并且不与Ceg1p相互作用。一个中间缺失的蛋白质Cet1(246 - 549)p，只有在过表达时才能支持细胞生长，主要以一种离散的盐稳定的11.5 S同型寡聚复合物形式沉降。这些数据表明，Cet1p从第230位氨基酸到第275位氨基酸的片段在调节自身缔合以及与Ceg1p结合方面发挥作用。遗传数据支持在Cet1p催化结构域两侧存在一个Ceg1p结合结构域，具体如下：(i) Ceg1p的过表达可抑制2μ CET1(246 - 549)的温度敏感型生长表型；(ii) Ceg1p的过表达可抑制温度敏感型丙氨酸簇突变CET1(201 - 549)/K250A - W251A；(iii) 其他15个在蛋白质其他位置发生错义变化的cet - 温度敏感型等位基因不能被Ceg1p的过表达所抑制。最后，我们表明，通过与小鼠加帽酶的鸟苷酸转移酶结构域融合，Cet1(275 - 549)p的体内功能得以完全恢复。我们推测，当三磷酸酶催化结构域通过与哺乳动物鸟苷酸转移酶顺式连接而被递送至RNA聚合酶II延伸复合物时，酵母RNA三磷酸酶对Ceg1p结合的需求可以被绕过。
