The Importance of Amino Acid Residues at the C-terminus of Region 1.1 to the Structural and Functional Properties of Bacilis subtilis sigA factor

中文摘要 因子是原核生物RNA聚合酶辨識啟動子DNA，並引發轉錄作用的重要蛋白。以往的研究結果顯示，Bacillus subtilis（枯草桿菌）A蛋白N-端保留性區域1刪除26，52，73，94，或103個胺基酸，均不影響A-RNA聚合酶之體外轉錄活性，但是刪除至106個胺基酸，A蛋白的轉錄活性就會急遽降低。為了探討A蛋白N-端第103個胺基酸至106個胺基酸對A蛋白功能之重要性，首先我分析A蛋白N-端第103至第106個胺基酸之順序性刪除，對A蛋白轉錄活性之影響。 實驗結果顯示，A蛋白N-端刪除104 或105個胺基酸，即可急遽降低突變型A-RNA聚合酶的轉錄活性。由於SND104-A蛋白僅缺乏A蛋白N-端的103個胺基酸，可見A蛋白N-端第103個胺基酸存在與否，對A蛋白能否保持正常的轉錄功能是相當重要的。以單循環體外轉錄作用分析野生型及刪除突變型A蛋白之轉錄特性，發現相較於野生型A-RNA聚合酶，SND103-A-RNA聚合酶具有轉錄延遲的現象。推測SND103-A-RNA聚合酶，至少可能在與啟動子DNA形成開放式複合體的效率上，已不同於野生型A-RNA聚合酶。另外，SND104-A-RNA聚合酶之體外轉錄缺失，更甚於SND103-A-RNA聚合酶。免疫沉澱、分子篩層析、以及膠體遲滯的研究結果顯示， SND104-A-RNA聚合酶轉錄活性明顯降低的的原因，和SND104-A蛋白與RNA聚合酶核心酵素之結合能力、或SND104-A-RNA聚合酶與啟動子DNA之結合能力無關。因此，我推測SND104-A-RNA聚合酶體外轉錄活性急遽下降低的原因，可能和其與啟動子DNA結合後，無法有效形成正常的開放式複合體，或更進一步進入轉錄延長反應有關。以圓二色光譜儀分析SND103-、SND104-、以及 SND105-A蛋白所保持的二級構造，結果顯示具有轉錄活性之SND103-A，比明顯具有轉錄缺失之SND104-、及 SND105-A蛋白，含有較少的二級構造。由於更多的刪除突變研究結果顯示，具有轉錄活性的SND100-、及SND102-A均比不具轉錄活性之SND104-、及 SND105-A，含有較少的二級構造，因此我認為A蛋白區域1.1及區域1.2交界處的胺基酸，對A蛋白能否擁有正確的功能構造，是相當重要的。Abstract The sigma (σ) subunit of prokaryotic RNA polymerase is essential for the recognition of promoter DNA and the initiation of transcription. Our previous data have revealed that the truncated A factor of B. subtilis, with N-terminal 26, 52, 73, 94, or 103 amino acids being deleted, is as functional as its wild-type counterpart after reconstitution with core RNA polymerase. However, further N-terminal deletion into residue 106 has resulted in an abrupt reduction in transcription activity of the RNA polymerase harboring the truncated A. This purpose of this study was to investigate the role of amino acid residues, spanning 103 to 106, on the structural and functional properties of A. The experimental data showed that both SND104- and SND105-A-RNA polymerases also significantly reduced their in vitro activities. Since the difference between SND103- and SND104-A was the presence or absence amino acid residue 103 at the N-terminus of the truncated A, it seemed that this amino acid was essential to the maintenance of functional A properties. Single cycle in vitro transcription has been adopted to compare the transcription properties of both the wild-type and N-terminally trancated A-RNA polymerases. The data revealed that SND103-A-RNA polymerase, in comparison with the wild-type one, had a delayed in vitro transcription activity, indicating that at least the efficiency of open complex formation was affected by removal of N-terminal 102 amino acids of A. The defect of delayed transcription was much more pronounced with SND104-A-RNA polymerase under the same condition. Reasons responsible for the defect of SND104-A-RNA polymerase were irrelevant to the core-binding activity of SND104-A and the promoter-binding activity of SND104-A-RNA polymerase as evidenced by gel filtration and gel retardation assays. It was probably a result of inefficient open complex formation of SND104-A-RNA polymerase and G3b promoter or a result of inefficient transition from transcription initiation to transcription elongation. To unravel the mystery resulting in the defect of SND104-A, I compared the amounts of secondary structures possessed by SND104- and SND105-A, which had reduced transcription activity, as well as SND103-A, which possessed normal multiple cycle transcription activity. Interestingly, data from circular dichroism showed that less amount of secondary structure was possessed by the active SND103-A than by the inactive ones. Since a relatively low amount of secondary structures was also observed for the active SND100- or SND102-A comparing with that for SND104-A, it tends to suggest that the amino acid residues located in the junction of regions 1.1 and 1.2 were very critical for the truncated A to possess a correct functional structure.目錄 壹、英文摘要……………………………………………………… 1 貳、中文摘要…………………………………………………….. 3 貳、前言…………………………………………………………… 5 參、材料與方法…………………………………………………… 12 一、試藥及實驗器材……………………………………………… 12 二、菌株與質體…………………………………………………… 12 三、實驗方法……………………………………………………… 12 1. pSNDX 刪除突變型sigA基因之構築……………………… 12 2. 刪除突變型σA蛋白之大量生產…………………………… 13 3. 刪除突變型σA蛋白之純化………………………………… 13 4. 純化具有histidine標示的枯草桿菌RNA聚合酶核心酵素 14 5. 蛋白質之定量………………….…………………………… 15 6. 刪除突變型σA蛋白的多循環體外轉錄活性 (in vitro multiple cycle transcription activity) 分析 ……………………………………………………………… 16 7. 刪除突變型σA蛋白的單循環體外轉錄活性 (in vitro single cycle transcription activity) 分析………………………….. 16 8. 刪除突變型σA蛋白的部分水解 (partial proteolysis) 分析… 17 9. 刪除突變型σA蛋白的二級結構分析……………………… 18 10. 刪除突變型σA蛋白與RNA聚合酶核心酵素結合能力之分析 (一)………………………………………………………………18 11. 刪除突變型σA蛋白與RNA聚合酶核心酵素結合能力之分析 (二)…………………………………………………………. 18 12. 枯草桿菌Φ29噬菌體G3b啟動子DNA之製備、純化及標示 …………………………………………………………………..19 13. 刪除突變型σA蛋白與Φ29噬菌體G3b啟動子DNA結合能力之分析………………………………………………………… 20 伍、結果…………………………………………………………… 21 一、刪除突變型σA蛋白基因之選殖與大量表現……………… 21 二、N-端刪除突變型A蛋白之純化………………………….. 21 三、B. subtilis β''次單元體具有His-tag標示之RNA聚合酶核心酵素的純化…………………………………………………....... .. 22 四、刪除突變型σA蛋白的體外轉錄活性(in vitro transcriptionactivity) 分析 …..…………………………………………….. 23 五、刪除突變型σA蛋白與core enzyme結合能力的分析………..23 六、刪除突變型σA蛋白與噬菌體G3b啟動子DNA結合能力 之分析…………………………………………………………..24 七、刪除突變型σA蛋白的單循環體外轉錄活性 (single cycle in vitro transcription activity) 分析…………………………………. 25 八、刪除突變型σA蛋白之構形分析………………………………26 九、SND100、102-σA蛋白的多循環體外轉錄活性 (multiple cycle in vitro transcription activity ) 分析………………………………27 陸、討論…………………………………………………………… 28 柒、附圖……………………………………………………………. 32 捌、附表…………………………………………………………… 44 玖、附錄........................................................................................... 45 拾、參考文獻...................................................................................... 5