McManus M T, Shimamura M, Grams J, Hajduk S L
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 35294, USA.
RNA. 2001 Feb;7(2):167-75. doi: 10.1017/s1355838201002072.
Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.
布氏锥虫的大多数线粒体基因不包含产生可翻译mRNA的必要信息。这些转录本必须经历RNA编辑，这是一个转录后过程，通过该过程，尿苷残基在线粒体mRNA中被添加和删除。RNA编辑被认为是由一种核糖核蛋白复合体催化的，该复合体含有内切核酸酶、末端尿苷酰转移酶（TUTase）、3'尿苷特异性外切核酸酶（U-exo）和连接酶活性。RNA编辑的催化酶均未被鉴定出来。在这里，我们描述了两种候选RNA连接酶（48 kDa和52 kDa）的鉴定，它们是布氏锥虫核糖核蛋白编辑复合体的核心催化成分。这两种酶与共价核苷酸转移酶超家族具有同源性，并包含五个关键的特征基序，包括活性位点KXXG。在本报告中，我们展示了关于拟议的48 kDa RNA编辑连接酶的数据。我们制备了针对重组48 kDa连接酶的多克隆抗体，该抗体能特异性识别锥虫酶。当在锥虫中作为表位标签融合蛋白表达时，重组连接酶定位于线粒体，与RNA编辑复合体结合，并与ATP发生腺苷酸化反应。这些发现为动基体RNA编辑的酶促级联模型提供了有力支持。
