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標題: 枯草桿菌RNA polymerase Sigma-A因子功能區域特性之探討
Identification and Characterization of the Functional Domains of Bacillus subtilis Sigma-A Factor
作者: 黃偉誠
Huang, Wei-Cheng
關鍵字: RNA 聚合;RNA polymerase;體外轉錄;Sigma-A factor;His-tag RNA polymerase;In vitro transcription;RNA polymerase core binding
出版社: 生物化學研究所
本研究旨在探討Bacillus subtilis sA蛋白上各個區域所具備有的特殊功能。實驗中我採用了七種刪除N-端及四種刪除C-端部份胺基酸序列的突變型sA蛋白,進行它們的生化特性分析。實驗結果顯示,含不同刪除突變sA蛋白之RNA聚合的體外轉錄活性,隨著胺基酸刪除之位置及胺基酸刪除數目的不同,而有明顯的差異。四種具有C-端刪除突變的sA蛋白,均喪失大部份的轉錄活性;在七種具N-端刪除突變的sA蛋白中,只有SND73-sA及SND94-sA和野生型sA一樣,具有明顯的轉錄活性。為了瞭解sA蛋白上各個位置的刪除對轉錄活性的影響機制,我分析了這十一種突變型sA蛋白與RNA聚合核心酵素結合的能力,以及含有這些突變型sA蛋白之RNA聚合與f29噬菌體G3b起動子DNA (promoter) 的結合能力。研究結果顯示,刪除突變型σA蛋白喪失轉錄活性的原因,大致可分為兩類,其中SND207-sA、SCD44-sA、及SCD79-sA等三個蛋白,因為無法與RNA聚合核心酵素結合,而導致它們轉錄活性的喪失。而SND26-sA、SND52-sA、及SND129-sA等突變種σA蛋白,則因為它們與核心酵素重組後所形成的RNA聚合無法與啟動子DNA結合所致。不同於上述兩類喪失活性的突變種σA因子,SND94-sA除了具有較野生型sA蛋白五倍高的核心酵素結合能力外,其所參與形成的RNA聚合亦比野生型sA所參與形成的RNA聚合,有1.5倍高的啟動子DNA結合能力。不過由於SND94-sA蛋白在RNA聚合與啟動子DNA之開啟式複合體形成效率上有所缺失,因此其轉錄活性僅及野生型sA蛋白50%。由上述實驗結果可推論,sA蛋白最C-端的胺基酸序列,對sA蛋白與核心酵素的結合能力有明顯的影響力;而sA蛋白最N-端的胺基酸序列,則決定sA-RNA聚合與啟動子DNA的結合能力,很可能sA蛋白N-端的26個胺基酸和sA蛋白在核心酵素中,進行正確的構形變化,使RNA聚合得以結合啟動子DNA的能力有關。另一個值得注意的事實是,雖然SND73-sA蛋白去除了sA蛋白最N-端的73個胺基酸,但是它仍具有和野生型sA蛋白相似的體外轉錄活性,以及轉錄起始所需的各種功能。藉此,我推測在B. subtilis菌體內此N-端73個胺基酸的存在,可能參與著sA蛋白所轉錄之基因的調控作用。

This study is aimed to explore the functional regions of the Bacillus subtilis sA factor. Seven mutants with deletion at the N-terminus and four mutants with deletion at the C-terminus of σA factor were studied for their biochemical properties. The in vitro transcription activity of the reconstituted RNA polymerase holoenzyme containing each of the truncated sA factors varied significantly, depending on the position and numbers of amino acid being deleted. All the four C-terminally truncated sA were inactive. Only SND73-sA and SND94-sA, among the seven mutants with deletion at the N-terminus, had transcription activities comparable to that of the wild-type counterpart after reconstitution with core RNA polymerase. Mechanisms leading transcription inactivation of the truncated sA-RNA polymerase can be categorized into two groups. SCD207-sA, SCD44-sA, and SCD79-sA belong to the first group, of which the activity loss was attributed to the inability of core association; whereas SND26-sA, SND52-sA, and SND129-sA belong to the second group, of which the reconstituted RNA polymerase holoenzymes have lost their promoter-binding activity. The SND94-sA factor had only 50% of the transcription activity of the wild-type counterpart. However, different from the properties of the above-mentioned two groups, it possesses a 5-fold higher activity in core association comparing with the wild type, and a 1.5-fold higher affinity for Φ29 phage G3b promoter after association with core. The decrease in abortive transcription activity of the SND94-sA-RNA polymerase suggests that it is inefficient in triggering the formation of open complex. Other important observations needed to be mentioned are that the most C-terminal part of sA plays an essential role in core binding and that the amino acids at the most N-terminal part of sA determine the promoter DNA-binding activity of the RNA polymerase holoenzyme. Probably, the N-terminal 26 amino acids would induce a conformational change of sA, which enables the holoenzyme to bind promoter DNA. Furthermore, the SND73-sA, although lacks the N-terminal 73 amino acids, remains active in vitro and possesses several essential functions required for transcription initiation. It seems that the N-terminal 73 amino acids of sA participate in the regulation of genes controlled by sA in B. subtilis.
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