Molecular Mechanism of tRNA Recognition by Aminoacyl-tRNA Synthetase from Hyperthermophilic Achaeon

超嗜热 Achaeon 氨酰 tRNA 合成酶识别 tRNA 的分子机制

基本信息

批准号：
16510154
负责人：
HASEGAWA Tsunemi
金额：
$ 2.65万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

项目摘要

To investigate the molecular recognition and discrimination mechanism of proline, tyrosine, histidine and tryptophan tRNA by each aminoacyl-tRNA synthetase (ARS) from hyperthermophilic and aerobic crenarchaeon, Aeropyrum pernix K1, each synthetase (ProRSAP, TyrRSAP, HisRSAP and TrpRSAP, respectively) gene was cloned, overexpressed and purified by using several purification procedures including affinity chromatography. Each amino acid specific wild type and mutant tRNA variants were prepared by in vitro transcription system with T7 RNA polymerase. These transcripts were aminoacylated with each amino acid by the recombinant ARS. ProRSAP recognized G35 and G36 of anticodon, discriminator base A73 and G1-C72 base pair at acceptor end. This G1-C72 specific recognition of acceptor end is different from Escherichia coli recognition site G72. This results suggest that the difference of recognition mechanism within A. pernix and E. coli ProRS may caused to change of nucleotides at position 1 an … More d 72 of proline tRNA during evolution. With respect to the long variable arm in E. coli tyrosine tRNA, some base pairs in length was required for tyrosylation. None of the recognition sites were found in the acceptor stem, except the discriminator base A73 in E. coli system. Archaeal and eukaryotic tyrosine tRNA have a unique C-G72 base pair. TyrRSAP did recognize this first C1-G72 pair in addition to the discriminator base A73 in the acceptor region and anticodon nucleotides. All known histidine tRNAs have an additional nucleotide at the 5' end compared with the other tRNA species. A. pernix histidine tRNA based on the information from database shows that A. pernix histidine tRNA does not have minus 1G residue. This additional minus 1G was strongly recognized by the HisRSAP. Surprisingly, minus IA and minus 1C histidine tRNA mutants also showed high histidylation activity. Discriminator base C73 was weekly recognized. HisRSAP does not recognize the anticodon similar to that of E. coli recognition system. HisRSAP can also recognize nonsense histidine tRNA. E. coli tryptophan tRNA is base-specifically recognized acceptor stem Al-U72, G2-C71 and G3-C70 base pairs, except anticodon nucleotides and discriminator base G73. TrpRSAP recognized all anticodon nucleotides C34, C35 and A36, discriminator base A73, G1-C72 and G2-C71 base pairs of acceptor stem. These results indicate that the recognition sites in the acceptor stem including discriminator base are so different between A. pernix and E. coli system. Less

To investigate the molecular recognition and discrimination mechanism of proline, tyrosine, histidine and tryptophan tRNA by each aminoacyl-tRNA synthetase (ARS) from hyperthermophilic and aerobic crenarcheon, Aeropyrum pernix K1, each synthetase (ProRSAP, TyrRSAP, HisRSAP and TrpRSAP, respectively) gene was cloned, overexpressed and purified by using几种纯化程序，包括亲和色谱。每种氨基酸特异性野生型和突变型TRNA变体都是通过T7 RNA聚合酶在体外转录系统中制备的。这些转录本通过重组ARs用每个氨基酸氨基酰化。 Prorsap在受体端识别了反密码子，歧视碱基A73和G1-C72碱基对的G35和G36。这种G1-C72对受体端的特异性识别与大肠杆菌识别现场G72不同。该结果表明，在A. pernix和大肠杆菌中，识别机制的差异可能导致位置1 an…更多d 72 proline tRNA的核动脉动物在进化过程中发生变化。关于大肠杆菌酪氨酸tRNA中的长可变臂，酪氨酸需要一些碱基对。除了大肠杆菌系统中的歧视剂A73以外，在受体词干中未发现任何识别位点。古细胞和真核酪氨酸tRNA具有独特的C-G72碱基对。除了在受体区域和反登核苷酸中的歧视碱基A73外，Tyrrsap确实认识了第一对C1-G72对。与其他TRNA物种相比，所有已知的组氨酸TRNA在5'端都有额外的核苷酸。 A.基于数据库的信息，pernix组氨酸tRNA表明，丙氨酸组氨酸tRNA没有负1G居住。 HISRSAP强烈认可了这一额外的负1G。令人惊讶的是，减去IA和减1C组氨酸tRNA突变体也显示出高的组酰化活性。每周认可判别者基础C73。 HISRSAP不认识到类似于大肠杆菌识别系统的反登基。 HISRSAP也可以识别胡疑的组氨酸tRNA。大肠杆菌色氨酸tRNA是基本识别的受体茎Al-U72，G2-C71和G3-C70碱基对，除了反登起核动肽和鉴别剂基碱基G73。 TRPRSAP识别了所有核动肽C34，C35和A36，歧视碱基A73，G1-C72和G2-C71碱基对的基础。这些结果表明，包括鉴别剂基库在内的受体茎中的识别位点在A. pernix和大肠杆菌系统之间是如此不同。较少的

项目成果

期刊论文数量（143）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

^1H, ^<13>C and ^<15>N chemical shift assignment of the C-terminal 15kDa domain of novel galactose-binding protein from the earthworm Lumbricus terrestris

来自蚯蚓的新型半乳糖结合蛋白的C末端15kDa结构域的^1H、^13C和^15N化学位移分配

DOI：
发表时间：
2004
期刊：
J. BiomolecularNMR 27(11)
影响因子：
0
作者：
Hemmi;H.;Kuno;A.;Hasegawa;T;et. al.
通讯作者：
et. al.

tRNA アイデンティティーからみた古細菌由来トリプトファニル-tRNA 合成酵素の分子進化

从tRNA身份角度研究古菌色氨酰-tRNA合成酶的分子进化

DOI：
发表时间：
2006
期刊：
影响因子：
0
作者：
Watanabe;T.;Hasegawa;T.;Fukuda K;et. al.;Yuji Hidaka;瀧真清・長谷川典巳・宍戸昌彦;Yuji Hidaka;福田宏太郎・西川富美子・西川論・長谷川典巳;Yuji Hidaka;菊池邦生・福田宏太郎・長谷川典巳;Masatoshi Saiki;渡部智也・長谷川典巳・福田宏太郎;Yuji Hidaka;Masatoshi Saiki;岡本幸司・久野敦・長谷川典巳;土屋渉・久野敦・長谷川典巳
通讯作者：
土屋渉・久野敦・長谷川典巳

The recognition mechanism of tryptophan tRNA by tryptophanyl-tRNA synthetase from thermophilic archaeon, Aeropyrum pernix K1.

嗜热古菌Aeropyrum pernix K1色氨酸tRNA合成酶对色氨酸tRNA的识别机制。