姓 名：逄爱萍 学 号： 日 期：

次级代谢产物是生物在特定条件下生成的具有重 要生理功能和活性的化合物。 次级代谢产物的结构是由其编码的基因簇决定的， 它的生成是基因协同表达的结果。 在链霉菌的次级代谢途径的合成基因簇中，功能 基因往往十几个以上，多达几十个，而调控基因 只有几个，甚至是一个。 背景

转录因子 原核生物转录 转录因子对靶基因启动子区域 DNA 序列的识别与结合是 整个调控的核心。 发现次级代谢途径特异性转录调控因子，深入研究其转 录调控机理，是微生物生理与代谢、生化与遗传的核心 关键问题之一。

Isolation of RNA RT-PCR Expression and purification of GST fusion proteins DNA-protein binding assays(EMSA) Footprinting assays Bioinformatic analysis 转录因子研究的一般方法

N C Monocistronic ：单顺反子，一个启动子后仅具有一个编码序列。 Polycistronic ：多顺反子，若干个基因由一个启动子控制，转录在一条 mRNA 上。 Organization of pimaricin gene cluster a tetraene macrolide antibiotic produced by S.natalensis

Primers ： 400——600bp S2S3 IS2 CG FS0 S3S4 JI GF Determining whether the neighboring genes could be co-transcribed by RT-PCR If unbated transcription was observed ， the neighboring genes were co-transcribed.

93aa 192aa 143aa Vector ： pGEX-2T Host: BL21 （ DE3) GST Construction of expression plasmids

Heterologous expression of PimM and of its truncated versions Expression ： 18 ℃， IPTG 0.1mM(OD=0.7) ， 14h Purification ： affinity chromatography on Glutathione sepharose

EMSA 原理： based on the separation of free DNA from DNA-protein complexes DIG Oligonucleotide 3’-End Labeling Kit, 2 nd Generation (Roche Applied Science) a.Digoxigenin labelled DNA b.DNA+protein incubation c.Electrophoresis:5% polyacrylamide native gel d.chemiluminiscene a d c b Determine the binding regions

DNA-protein binding assays ： EMSA One retarded bands: pimKp, pimAp, pimEp, pimS2p,pimIp; Two retarded bands: pimS1-Dp; Four retarded bands: pimJp; Two negative control reactions: absence of protein, and use of GST Left lane: control without protein Right lane: 60 uM of GST-PimM protein

B:a competition experiment between pimJp and pimCp. Addition of one to 1000-fold higher concentrations of pimCp competitor DNA failed to diminish the intensities of the pimJp retardation bands C:control reactions made with pure GST protein were negative in all cases, excluding a possible binding of this protein to the promoters

B.PAS domain reduces binding affinity Figure A demonstrates that binding ability relies on the DNA-binding domain, and is independent of the PAS domain. Figure B demonstrates that truncated forms of the protein have significantly higher affinity. A. binding ability relies on the DNA-binding domain

Dnase I 足迹实验 （ footprinting assay ） a.6-FAM labelled DNA +protein incubation b.Dnase I digestions c.Analyse with PEAK SCANNER program Determine the binding sequence

The retarded band was observed upon the incubation of GST-PimM with all the promoters which are similar to PimM binding site. A,B,C :homologous regulators from different polyene producers The figure suggest the orthologous regulators of polyene biosynthesis share the same regulatory pattern

Genetic complementation of S.natalensis ΔpimM by orthologous regulators restores pimaricin production DNA fragment: pimM,amphRIV, nysRIV, pteF Vector: pSET152 giving rise to pSETpimM pMamphRIV, pMnysRIV, pMpteF Transfermation by conjugation As expected,given its highest identity to PimM, pimaricin yield was the highest in the strain complemented with pteF (94%) Production of the strain complemented with amphRIV or nysRIV, which are more distantly related to pimM, was somewhat lower, (47%,61%)

When we introduce one copy of pimM into the genomes of S.nodosus and S.avermitilis, the polyene production boosted substantially, whereas no significant change in the growth curve was observed. Expression of PAS-LuxR regulators is a bottleneck Confirm the functional conservation among polyene biosynthetic gene clusters.