Transcript 发现次级代谢途径特异性转录调控因子

姓
名：逄爱萍
学
号：2012207355
日
期：2013.09.13
背景
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。
原核生物转录
转录因子
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。
转录因子研究的一般方法
Isolation of RNA
RT-PCR
Expression and purification of GST fusion
proteins
DNA-protein binding assays(EMSA)
Footprinting assays
Bioinformatic analysis
Organization of pimaricin gene cluster
a tetraene macrolide antibiotic produced by S.natalensis
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。
N
C
Determining whether the neighboring genes could
be co-transcribed by RT-PCR
Primers： 400——600bp
S2S3
S3S4
IS2
JI
CG FS0
GF
If unbated transcription was observed，the neighboring genes
were co-transcribed.
Construction of expression plasmids
192aa
GST
143aa
93aa
Vector：pGEX-2T
Host:
BL21（DE3)
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：
a
Determine the binding regions
b
DIG Oligonucleotide 3’-End
Labeling Kit, 2nd Generation
(Roche Applied Science)
c
a.Digoxigenin labelled DNA
b.DNA+protein incubation
c.Electrophoresis:5%
polyacrylamide native gel
d.chemiluminiscene
d
based on the separation of
free DNA from DNA-protein
complexes
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
A. binding ability relies on the DNA-binding domain
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.
B.PAS domain reduces binding affinity
Dnase I足迹实验
（footprinting assay）
Determine the binding
sequence
a.6-FAM labelled DNA
+protein incubation
b.Dnase I digestions
c.Analyse with PEAK
SCANNER program
A,B,C :homologous regulators
from different polyene
producers
The retarded band was
observed upon the incubation
of GST-PimM with all the
promoters which are similar to
PimM binding site.
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%)
Expression of PAS-LuxR regulators is a bottleneck
Confirm the functional
conservation among
polyene biosynthetic gene
clusters.
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.