RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates
RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates
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DOI：
10.1073/pnas.1201207109
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发表时间：
2012-04-17
影响因子：
11.1
通讯作者：
Shuman, Stewart
中科院分区：
文献类型：
--
作者：
Chakravarty, Anupam K.;Subbotin, Roman;Shuman, Stewart
核酸酶学的一个宝贵原则认为，多核苷酸3‘-5’磷酸二酯的合成是通过3‘- oh对高能5’磷酸酐的攻击进行的：对于RNA/DNA聚合酶来说，是核苷5'-三磷酸，对于多核苷酸连接酶来说，是腺苷化的中间物A(5')pp(5')N-。RtcB是一个涉及tRNA剪接和修复的RNA连接酶家族的例子。与传统的连接酶不同，RtcB用3'-磷酸和5'-OH末端密封断裂的rna。在这里，我们发现RtcB执行一个三步连接途径，包括(i)酶的His337与GTP反应形成共价的RtcB-（组氨酸- n）- gmp中间体；（ii）鸟苷转移到多核苷酸3′-磷酸，形成多核苷酸-（3′）pp（5′）G中间体；（iii） 5′-OH对-N（3′）pp（5′）G端的攻击，形成拼接结。RtcB具有独特的结构和化学机理。RtcB蛋白在细菌、古生菌和后生动物中的广泛分布，提出了基于共价激活的3′端替代酶学的前景。
A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3'-5' phosphodiesters proceeds via the attack of a 3'-OH on a high-energy 5' phosphoanhydride: either a nucleoside 5'-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5')pp(5')N- in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3'-phosphate and 5'-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3'-phosphate to form a polynucleotide-(3')pp(5')G intermediate; and (iii) attack of a 5'-OH on the -N(3')pp(5')G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3' ends.