Study On The Structure And Function Of Human Mitochondrial TRNA～(Leu) In Vitro Recombination Of The Two Subunits Of Leucyl-tRNA Synthetase From Aquifex Aeolicus

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.