Gene Expression and Regulation of Bioluminescence: Functional Analysis of the Regulatory Gene of the lux Regulon form Vibrio fischeri

Abstract

本論文為了更深入探討螢光的調節機制及減少變因,只取主要的調控區域 luxR-R&R-luxI 作為研究對象,分別針對luxR 和 luxI 基因之產物及基因 之產物及基因本身之 DNA 序列所具有的功能及其內在意義等主題加以探 討。在此設計了一系列調節基因次序重組之質體及 in trans互補質體,利 用 V. harveyi 之螢光.blksq.基因作為報導基因,並於前後各插入 CaMV35s promoter 及 NOS-terminator,以避免來自質體中其它 promoter 的干擾;藉由不同組合之質體的螢光表現情形,對luxR 和 luxI 基因的產物及其 DNA 序列在調節機制中所扮演的角色有更深入的瞭解。 實驗結果顯示 LuxR 和 autoinducer 必需同時存在才具有功能,且其作用 部位在調節區域中,此結果印証並提供 LuxR 和 autoinducer 會形成 LuxR-AI 複合體之間接証據。此外,亦發現有 LuxR 或 LuxR-AI 複合體存 在時,luxR 基因之 DNA 序列對於 luxR 的表現有明顯的抑制作用。故推 測 luxR 基因上可能有一個 LuxR motif-binding locus,藉此進行 negative feedback control 調控 L-operon 的表現。分析 luxR gene 之 DNA 序列,於其 N-terminal 發現一個對稱性序列 R3-reversed re- peat,其序列為 TATTTACTCGCGATCATTTAT, 因而利用定點突變來確認此序 列是否為 LuxR 之作用部位。利用 insertion 造成 frame-shift 或直接 突變產生 stop codon 使轉譯提前終止,結果使 L-operon 的表現大幅度 降低,顯示 luxR 基因之 mRNA 可能具有形成 2°級結構的潛力,而產生類 似attenuation 的作用,而抑制 L-operon 的表現;in trans 加入 LuxR- AI 複合體僅有些微的作用,與 wild type 相比仍相距甚遠,由此可知所形 成之 2°級結構具有相當的穩定性;分析 luxR 之 mRNA 的結構,此亦顯示 其具有形成 2°級結構之 potential。而不改變 LuxR 之胺基酸序列,僅 於 DNA 序列上作改變的結果,無法肯定証實 R3-reversed re- peat 為 LuxR negative feedback control 的作用部位;可能 LuxR 的 negative feedback control 屬於一種弱勢調控作用,其調節機制類似 attenuation 的作用,以阻緩 translation 來終止 transcription 而抑 制基因的表現,真正的機制仍需進一步研究。

For the purpose of simplicity, only the regulatory genes, luxR and luxI, were used for studying in this work. A series of gene order rearrangement constructions and in trans comple- mentary tests were designed to investigate the requirement of the sequences of the luxR and luxI genes, and functional analy- sis of the LuxR protein and autoinducer (AI). The luciferase genes, luxA-luxB, from V. harveyi were used as reporter gene to monitor the expression of the R- and L-promoter. The results show that the sequences of the luxR and luxI genes were the ne- gative control elements for the expression of the R- and L-pro- moter. The LuxR-AI complex is the positive control element for both the R- and L-promoter, and directly bound to the R&R se- quence. The sequence of the luxR gene repressed the expression of the L-promoter much stronger than the effect of the sequence of the luxI gene to the R-promoter. Nucleotide sequence analysis showed that an reversed repeat sequence, termed R3- reversed re- peat, was found on the sequence of the luxR gene, and it might be the LuxR motif-binding locus. The site-directed mutagenesis was used to inspect the function of this site. The results of the luxR mutations show that the premature translation of the LuxR protein repressed the expression of the following reporter genes. It suggested that the sequence of the luxR gene might re- gulate the lux regulon by attenuation-like termination. Further modification in this site was be done to define the function of the R3-reversed repeat, but the result can't confirm R3-reversed repeat is LuxR protein motif-binding locus for negative feedback control. The LuxR protein might bind on one specific site of the luxR sequence to enhance the attenuation-like transcriptional termination and negative feedback control the expression of L- operon.