Exchange of regions between bacterial poly(A) polymerase and the CCA-Adding enzyme generates altered specificities
Exchange of regions between bacterial poly(A) polymerase and the CCA-Adding enzyme generates altered specificities
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DOI：
10.1016/j.molcel.2004.06.026
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发表时间：
2004-08-13
期刊：
影响因子：
16
通讯作者：
Mörl, M
中科院分区：
文献类型：
--
作者：
Betat, H;Rammelt, C;Mörl, M
细菌聚(A)聚合酶（PAP）和tRNA核苷酸转移酶在序列上高度相似，但表现出不同的活性：tRNA核苷酸转移酶催化在tRNA的3'端添加CCA，而PAP在多种转录本中添加聚(A)尾部。通过结构域替换实验，我们发现这些酶遵循模块化概念：N端和c端区域的交换导致嵌合酶具有意想不到的活性，表明tRNA核苷酸转移酶在c端部分携带“锚定结构域”，限制聚合为三个核苷酸。鉴定了一个27个氨基酸区域，决定了聚(A)或CCA是由酶嵌合体合成的。序列比对表明，这两种酶的催化核心携带参与核苷酸识别和结合的相同成分。这似乎是观察到的PAP催化中心重编程的先决条件，以纳入确定长度和组成的序列，而不是长链的a残基。
Bacterial poly(A) polymerases (PAP) and tRNA nucleotidyltransferases are highly similar in sequence but display different activities: whereas tRNA nucleotidyltransferase catalyzes the addition of CCA to 3' ends of tRNAs, PAP adds poly(A) tails to a variety of transcripts. Using domain substitution experiments, we show that these enzymes follow a modular concept: exchange of N- and C-terminal regions leads to chimeric enzymes with unexpected activities, indicating that tRNA nucleotidyltransferase carries an "anchor domain" in the C-terminal section that restricts polymerization to three nucleotides. A 27 amino acid region was identified that determines whether poly(A) or CCA is synthesized by the enzyme chimeras. Sequence alignments suggest that the catalytic cores of both enzymes carry identical components involved in nucleotide recognition and incorporation. This seems to be the prerequisite for the observed reprogramming of the catalytic center of PAP to incorporate a sequence of defined length and composition instead of long stretches of A residues.