Sigma 54 是細菌體內一種次要的轉錄因子，與主要的轉錄因子 sigma 70 不同，但是其轉錄機制與真核生物的 RNA 聚合 II 所產主導轉錄機制很像，包括都需要 ATP 的水解，才能使雙股 DNA 的分離；都需要活化子才能促進轉錄的起始；與 RNA 聚合 II 轉錄因子相似的特色結構。先前研究將 sigma 54 的 N 端白胺酸七重複區域與區域 III 中疏水性七重複截斷，會影響sigma 在glnAp2啟動子-12 區域的結合。經由突變點證實在區域 I 疏水性七重複是辨認啟動子-12 區域及轉錄活化的重要位置。為了分析 sigma 54區域 III 疏水性-酸性區域上疏水性殘基對轉錄的影響，我們將區域 III 疏水性七重複區域利用點突變將白胺酸或異白胺酸改變為親水性絲胺酸，建立一系列單點突變、雙點突變及三點突變，並進而以硫酸二甲基足跡法(DMS footprinting)，高錳酸鉀足跡法(permanganate footprinting)，核心結合測試(core-binding assay)及 mRNA 產量分析(transcription assay)的方法來分析突變的sigma 54 對轉錄的影響。
由高錳酸鉀足跡法結果顯示在單點突變及雙點突變的 sigma 54 ，並不會影響啟動子和 RNA 聚合複合體之張開式複合體的形成而在三點突變的 sigma 54幾乎完全被破壞，而無法形成張開式複合體。由硫酸二甲基足跡法的結果顯示雙點突變sigma 54 只有少部分結合到 glnAp2 啟動子-12 區域上，而三點突變 sigma 54 幾乎不能結合到-12 區域。另外，在雙點突變 sigma 54轉錄製造 mRNA 的能力下降至原型 sigma 54 之30%至40%，三點突變 sigma 54則下降至原型之20%或小於10%。在sigma 54 與核心酵素結合能力分析中，發現雙點突變 sigms 54 與核心酵素結合能力下降，三點突變 sigma 54 與核心酵素結合能力完全喪失。由以上的結果，顯示此區域上疏水性胺基酸的破壞將破壞sigma 54 和核心的結合能力，進而導致與啟動子-12 區域的辨認對轉錄起始作用的下降。因此，本實驗證實了 sigma 54 區域 III 疏水性七重複區域的疏水性殘基是核心結合的重要的區域。
Escherichia coli transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich. Previous deletion studies suggested that the leucine heptad repeats in the N-terminal region may work together with recongnition on ─12 region. It was demonstrated through site-directed mutagenesis study that hydrophobic residues on the heptad repeats in Region I are necessary for ─12 promoter element recongnition and transcription activation. In order to specify the roles of hydrophobic residues in Region III, we performed site-directed mutagenesis to create mutation in the leucines on the hydrophobic heptad repeats. A series of mutants with single, double and triple mutations have been created and characterized by the use of in vivo permanganate footprinting, dimethyl sulfate (DMS) footprinting, core binding and transcription assays.
The results of permanganate footprinting indicated that single- or double-amino-acid substitutions showed no discernible loss of transcription start opening. Nevertheless, the two triple mutants almost abolished the transcription start site opening. On the other hand, double mutants showed only partial abilities to bind core RNA polymerase and partial protection on the －12 promoter element of glnAp2 promoter. The triple mutants lost most, if not all, of their ability to protect the －12 promoter element in the DMS in vivo assays, and their ability to bind core RNA polymerase. In addition, double mutations are deleterious to the function of sigma 54, indicated by retaining only 30%-40% of the wild type mRNA level. Two triple mutants produced about 20% or less than 10% of the wild type transcripts in the glnAp2 promoter. Our results suggest that the hydrophobic residues of the heptad repeats of Region III are important for the function of core RNA polymerase binding. Here we conclude that a progressive loss of the hydrophobicity on the hydrophobic heptad repeats in Region III of sigma 54 result in a progressive loss of core-binding ability, recognition of the －12 promoter element and mRNA production.
