【作者】 郝睿；

【导师】 王恩多；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2006， 博士

【摘要】 线粒体性脑肌病,乳酸中毒和中风样发作综合症（MELAS症）是一种罕见的先天性线粒体DNA病,有报道指出,位于编码人线粒体tRNA^Leu（UUR）基因中的五种点突变与MELAS症相关。我们构建并转录获得了这些tRNA突变体,通过体外的氨基酰化实验,测定了这些点突变对tRNA接受茎活力的影响。结果显示,和野生型tRNA相比,带有这五种点突变的tRNA^Leu（UUR）转录子的氨基酰化水平都有不同程度的降低。进一步的热稳定性实验证明,氨基酰化水平最低的A3243G突变体和T3291C突变体的结构不稳定。另外,T3291C突变体能够竞争性地抑制野生型tRNA的亮氨酰化,暗示携带该突变的tRNA可能作为一种抑制剂存在于患者的线粒体内。人线粒体亮氨酸tRNA的CUN等受体(hmtRNA^Leu（CUN）)负责解码线粒体蛋白质编码系统中使用频率最高的密码子,目前有六个致病性点突变被定位于它的基因内部。本文的体外实验显示,和野生型的hmtRNA^Leu（CUN）转录子更多还原

【Abstract】 The mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-likeepisodes syndrome （MELAS） is a rare congenital disorder of mitochondrial DNA（mtDNA）. Five single nucleotide substitutions within the human mitochondrialtRNA^Leu（UUR） gene have been reported to be associated with MELAS. Here weprovide in vitro evidence that the aminoacylation capacities of these fivehmtRNALeu（UUR） transcripts are reduced to different extents relative to thewild-type hmtRNA^Leu（UUR） transcript. A thermal denaturation experiment showedthat the A3243G and T3291C mutants, which were the least charged by LeuRS, havefragile structures. In addition, the T3291C mutant can inhibit aminoacylation of thewild-type hmtRNA^Leu（UUR）, indicating that it may act as an inhibitor in themitochondrial heteroplasmic environment.The human mitochondrial tRNALeu（CUN） （hmtRNALeu（CUN）） corresponds tothe most abundant codon for leucine in human mitochondrial protein genes. Here, invitro studies reveal that the U48C substitution in hmtRNALeu（CUN）, whichcorresponds to the pathological T12311C gene mutation, improved theaminoacylation efficiency of hmtRNALeu（CUN）. Enzymatic probing suggested amore flexible secondary structure in the wild type hmtRNALeu（CUN） transcriptcompared to the U48C mutant. Structural analysis revealed that the flexibility ofhmtRNALeu（CUN） facilitates a T-stem slip resulting in 2 potential tertiary structures.Several rationally designed tRNALeu（CUN） mutants were generated to examine thestructural and functional consequences of the T-stem slip. Examination of thesehmtRNALeu（CUN） mutants indicated that the T-stem slip could change the tRNAtertiary structure as well as govern its charging activity. These results suggest anovel, self-regulation mechanism of tRNA structure and function. Different tRNAtranscripts were bound on beads respectively, by which we screened tRNA bindingprotein from human mitochondria. Using this method, we found an unknown proteinthat can recognize specific tRNA structure. This result cast a new light on themechanism of related mitochondrial disease.The Aquifex aeolicus αβ-LeuRS is the only known heterodimericLeucyl-tRNA synthetase. The His6-tagged α-subunit gene was cloned into anexpression vector, which was transferred into E. coli BL21（DE3）. After lowtemperature induced expression, the soluble His-α protein was purified in a singlestep by metal （Ni2+） chelate affinity chromatography. We mixed the His-α and β invitro and found that the recombinant protein recovered leucylation activity.Compared with several other factors, urea promotes recombinant efficiencyobviously.更多还原