第四届九源奖学金一等奖论文摘要（1）

1、Cloning, characterization and mapping of the human ATP5E gene, identification of pseudogene ATP5EP1, and definition of the ATP5E motif（发表刊物：Biochem. J. (2000) 347,17-21(Printed in Great Britain）

A cDNA encoding the ε subunit of human ATP synthase, ATP5E, was isolated from heart, skeletal muscle and spleen cDNA libraries respectively, Its genome structure was characterized as comprising three exons and two introns within a stretch of 5 kb, according to the genomic sequence AL109840. The gene was mapped to human chromosome 20q13.3 between marker D20S173 and 20qter using the radiation hybrid GB4 panel. Northern blot analysis showed that the ATP5E gene was expressed as a single 0.6kb transcript in all 16 human tissues tested, with a high level present in heart and skeletal muscle. A new conserved motif composed of 24 residues, termed the ATP5E motif [W(R/K)X5YX2(Y/F)X3(C/A)X4RX3K], was defined on the basis of sequence of ATP synthase ε subunits from ten different organisms. In addition, a pseudogene ATP5E was also identified on the basis of genomic sequence AC004066, localized o9n human chromosome 4q25. By analyzing these results combined with the Southern blot patterns of human DNA hybridized with bovine ATP5E cDNA repotted previously [Vinas, Powell, Runswick, Iacobazzi and Walker (1990) Biochem. J.265, 321-326], we provide evidence of yet further homologous sequence (either gene or pseudogene) of ATP5E, in addition to ATP5E and ATP5E and ATP5EP1 in the human genome.

作者：屠强(中科院生化细胞所，生物化学与分子生物学博士，导师：余龙教授)

2、Trimeric ring-like structure of ArsA ATPase（发表刊物：FEBS Letters 469 (2000) 105-110）

ArsA protein is the soluble subunit of the Ars anion pump in the Escherichia coli membranewhich extrudes arsenite or antimonite from the cytoplasm .The molecular weight of the subunit is 63 kDa. In the cell it hydrolyzes ATP, and the energy released is used by the membrane-bound subunit ArsB to transport the substrates across the membrane. We have obtained two-dimensional crystals of ArsA in the presence of arsenite on negatively-charged lipid monolayer composed of DMPS and DOPC. These crystals have been studied using electron microscopy of negatively-stained specimens followed by image processing. The projection map obtained at 2.4 nm resolution reveals a ring-like structure with threefold symmetry. Many molecular assemblies with the same ring-shape and dimensions were also seen dispersed on electron microscopy grids, prepared directly from purified ArsA protein solution. Size-exclusion chromatography of the protein sample with arsenite present revealed that the majority of the protein particles in solution have a molecular weight of about 180 kDa. Based on these experiments, we conclude that in solution the ArsA ATPase with substrate bound is mainly in a trimeric form.

作者：王宏伟（清华大学生物科学与技术系，生物物理专业博士，导师：隋森芳教授）

3、CP1 Domain in Escherichia coil Leucyl-tRNA Synthetase is Crucial for its Editing Function（发表刊物：Biochemistry 2000,39,6726-6731）

The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. In this study, we created LeuRS-B, a mutant leucyl-RNA synthetase from Escherichia coil with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain), This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNALeu by methionine or isoleucine, suggesting that it is impaired the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNALeu made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-RNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucuyl-tRNA synthetase to fulfill its editing function.

作者：陈剑峰（中国科学院上海生物化学研究所，生物化学博士，导师：王恩多研究员）

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