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研究生:邱世昌
論文名稱:預測IS1351的C端核心催化區的分子模型
論文名稱(外文):Predicting structural model of the catalytic core domain of the insertion sequence ISLC3 from Lactobacillus casei ATCC 393
指導教授:林志侯
指導教授(外文):T. H. Lin
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:56
中文關鍵詞:乳酸菌轉位子分子動力模擬
外文關鍵詞:Lactobacillus caseiMolecular DynamicsTransposon
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乳酸菌 Lactobacillus casei 中所分離出來的 ISLC3(AF445084) 為轉位子 IS3 family 成員之一,長1,351 bp,可轉譯 382 個胺基酸。ISLC3 包含兩個開放編譯框 (open reading frame)。IS3 family 見存於在革蘭氏陽性菌 G(+)與革蘭氏陰性菌 G(-)中約40種細菌,並以非複製形態進行轉位機制。轉位子是很好的基因突變體,轉位子與基因體 DNA 重組或質體嵌入基因體息息相關,具有重塑基因庫的能力,在基因遺傳和演化上,扮演極為重要的角色。但是太頻繁的轉位作用所伴隨產生的基因組重組現象會導致細胞的損傷,因此轉位作用的調控極為重要。反轉錄病毒(retrovirus)的 Integrase (核酸銜接酵素)所扮演的角色是將反轉錄酵素所反轉錄之 cDNA 銜接嵌入至寄主細胞的標的 DNA (target DNA)基因組中,在銜接嵌入過程中也有著類似的轉位機轉。
對整個 IS3 family而言,N 端在胺基酸序列上較不保守,但均有 helix-turn-helix DNA binding domain ; C 端有一個核心催化區 (catalytic core domain),含有活化催化位置 DDE motif的結構,負責轉位機制(transposition)過程中 DNA 的銜接/瓦解的所有催化反應。其催化反應中心具有 Asp 及 Glu 兩個殘基 (residue),可以與二價金屬離子結合配位,維持催化中心的活性。本篇研究利用了電腦模擬的方法,利用現有的理論、資料庫及軟體(InsightII),建立 ISLC3 的 C 端核心催化區的分子模型,建立分子模型的資料,將有助於分子結構和其功能關係之研究,對 ISLC3 轉位子分子機制將能有更深入的瞭解。

ISLC3 is a member of IS3 family that is isolated from Lactobacillus casei. It has 1,351 bp and encodes for 382 amino acids. ISLC3 contains two open reading frames, named orfA and orfB, respectively. IS3 family can be found in both Gram positive and Gram negative bacteria genomes, and it replicates in a no conservative way. Transposon is a good mutagen that is related to DNA recombination or insertion of plasmid DNA into genome. Since it has an ability of reshaping the genome, it plays a critical role in evolutionary genetics. However, the recombination of genome with high frequent transposition can be a harm to the host, so the regulation of transposition is necessary. The integrase of retrovirus plays a role in insertion of reverse-transcripted cDNA into the target DNA in host genome,
and the process of insertion is similar to transposition.
The N terminal of IS3 family is relatively nonconserved, but all contain helix-turn-helix DNA binding domain. The catalytic core domain in C terminal contains the active site, DDE motif, function in linkage/terminal all catalytic reactions. The two residues, Asp and Glu, in the catalytic site are coupled to a divalent metal ion that is essential to its activity. I used a computer stimulation method with current theory, database and software InsightII, to constructed the molecular model of C terminal catalytic core domain of ISLC3. The molecular model of ISLC3 helps to study on the relationship between molecular structure and function in order to understand
the mechanism of ISLC3 transposition.

目 錄
摘要………….…………………………..………………………………1
Abstract………….…………………………..…………………………2
第一章、簡介………….…………………………..…………………..3
1-1 ISLC3 簡介………………. ….……………..…………………...3
1-2 預測蛋白質結構的方法…………………………………………..6
1-3 蛋白質分子結構的特徵………..………………………………...8
1-4 分子動力模擬(MolecularDynamics ,MD ) …………..…….….10
第二章 實驗原理和方法…..……..………………………………..12
2-1 同源模擬法預測蛋白質分子結構……………………..…….….12
2-2 InsightII 軟體……..…………………………………...…..….16
2-3 模擬與二價金屬離子結合配位……………..…………………...18
2-4 分子動力模擬原理………………………………………………..19
第三章 結果……..…….………..…….…………………………….…29
第四章 討論……..….…………………………………………………45
第五章 參考資料 ……………………………………………………46
附錄一 HFR、HMM 模版結果……... ..……………………………………53
表目
表、一 由HFR篩選出的模版資料………………………………………29
表、二 由HMM篩選出的模版資料 ………………………………………30
表、三 ISLC3的核心催化區對模版的序列百分比 ……………………31
表、四 模版所 Model 出結構的RMSD值比較 …………………………31
表、五 ISLC3 結構Free Mg2+及Mg2+結構的構形能比較………………37
表、六 D1D2E motif,D1各原子與二價金屬離子的距離(Å)…………42
表、七 D1D2E motif,D2各原子與二價金屬離子的距離(Å)…………43
表、八 D1D2E motif,E各原子與二價金屬離子的距離(Å)…………44
圖目
圖、一 1asu、1bi4C、1bisB、1c0m、1bl3C及2itg 經由 SWISS-MODEL
Protein Modelling Server 所預測出的結構………………33
圖、二(A)ISLC3 的 C 端核心催化區與模版的序列比對……………34
(B)為紅線區為所預測出結構的序列部分 ……………………34
圖、三ISLC3 核心催化區結構與ISLC3 結構的Free Mg2+和Mg2+結構立體圖……………………………………………………………36
圖、四Hiv-1 Integrase Core Domain Complexed With Mg2+(1BIU),DDE motif 各原子與Mg2+的距離(Å)………………………………38
圖、五Crystal Structure Of Tn5 Transposase Complexed With Mn2+(1F3I),DDE motif 各原子與Mn2+的距離(Å)…………………39
圖、六Catalytic core domain of ISLC3 Complexed With Mg2+,DDE motif 各原子與Mg2+的距離(Å)………………………………40
圖、七DDE motif,各胺基酸分子的原子名稱 …………………………41

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