第八章真核基因表达调控(1)

1. RNA polymerase II 2. promoter and enhancers 3. transcription factors
Ordered Assembly and Pol II Holoenzyme
one-step
multiple-step
TFIID TFIID
Sequential Assembly
tat protein of HIV can stimulate transcription initiation without binding DNA at all
The activating domain of the tat protein can stimulate transcription if it is tethered in the vicinity of promoter by binding to the RNA product (tar sequence) of a previous round of transcription.
(一) RNA聚合酶
RNA Pol I: rRNA, 相对活性50-70% RNA Pol II: mRNA,相对活性20-40% RNA Pol III: tRNA,相对活性10% RNA Pol IV: small ncRNA,相对活性??
（二）特异DNA序列
真核生物基因组中含有可以调控自身基因表达活性的特异DNA 序列，称为顺式作用元件 (cis-acting element)。
是指能够直接或间接与启动子核心序列TATA盒特异结合、 并启动转录的一类调节蛋白。
holoenzyme
TAF: TBP associated factors
TFⅡF polⅡ
TBP: TATA-box binding protein
TAF TAF
TFⅡA
TAF TFⅡH
TFII: pol II associated TF
能直接或间接与顺式作用元件相互作用，进而调控基 因转录的一类调节蛋白，统称为反式作用因子。
按其功能不同，常有以下三类：
基本转录因子 :识别promoter元件
转录调节因子:识别enhancer或silencer
共调节因子：不能进行DNA-蛋白质相互作用
1. 基本转录因子 (general transcription factor, GTF)
Mediator complex is targeted by an activator (中介复合体）
Far
• Mediator is a stable complex containing several proteins (20-50) • Mediator binds to the RNA pol II and transcription factors (activators or repressors) and ‘mediates’ the regulatory signals to pol II
一、真核基因表达的时间性及空间性
（一）时间特异性
按功能需要，某一特定基因的表达严格按
特定的时间顺序发生，称之为基因表达的时间
特异性(temporal specificity)。
多细胞生物基因表达的时间特异性又称阶
段特异性(stage specificity)。
Hale Waihona Puke Baidu
人体发育过程中不同类型β -珠蛋白的含量变化
DNA
TBP
TFⅡB
TATA
RNA聚合酶Ⅱ在转录因子帮助下，形成的转录起始复合物
2. 转录调节因子 (transcription factor, TF)
这类调节蛋白能识别并结合转录起始点的上游序列和远 端的增强子元件，通过DNA－蛋白质相互作用而调节转录活 性。决定不同基因的时间、空间特异性表达. 转录激活因子(transcriptional activator)
2） TF常见的trans-activation domain
(Activation domain is interchangeable)
Interaction Assays
Design of Two-hybrid / Three-hybrid /etc…
separable functional domains
Two-hybrid assay (protein-protein)
Tri-hybrid assay (protein-RNA)
真核基因转录起始的调控
Eukaryotic gene expression is usually controlled at the level of initiation of transcription.
· TFIIE and TFIIH are required to melt DNA to allow polymerase movement. · Phosphorylation of the CTD (by TFIIH and other kinases) is required for elongation to begin—fire the Pol II. · The CTD may coordinate processing of RNA with transcription.
转录阻遏因子(transcriptional repressor) 3. 共调节因子 (transcriptional regulator/ co-factor) 首先与转录因子发生蛋白－蛋白相互作用，进而影响它 们的分子构象，以调节转录活性,本身无DNA结合活性。 如果与转录激活因子有协同作用——共激活因子； 与转录阻遏因子有协同作用——共阻遏因子。
顺式作用元件能够被转录调节蛋白特异识别和结合，从而影
响基因表达活性。 启动子 (promoter) 顺式作用元件又分 增强子 (enhancer) 沉默子 (silencer)
DNA
转录起始点
En/Si
Pro
编码序列
（三）真核基因的调节调节蛋白
反式作用因子 (trans-acting factor)
蛋白质-蛋白质结合域
（dimerization, co-factors）
1） TF最常见的DNA binding domain
Zinc Finger bZIP
Homeodomain
bHLH
(1) 锌指(zinc finger)
Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His
tat
tar
DNA-binding domain is to bring the activation domain into the vicinity of the startpoint. And activation is independent of the means of tethering.
（二）空间特异性
在个体生长全过程，某种基因产物在个体 按不同组织空间顺序出现，称之为基因表达的
空间特异性(spatial specificity)。
基因表达伴随时间顺序所表现出的这种分 布差异，实际上是由细胞在器官的分布决定的， 所以空间特异性又称细胞或组织特异性(cell or tissue specificity)。
C-terminal: α-helix binding DNA
常结合GC box
Cys-X2-Cys-X13-Cys-X2-Cys
(2) 碱性亮氨酸拉链 bZIP
碱性亮氨酸拉链（bZIP: basic Leu Zip)结构域转 录激活因子序列比较
(3) 碱性螺旋-环-螺旋bHLH
bHLH蛋白(basic Helix-Loop-Helix)
第八章 真核基因表达调控
黄红辉 honghuih@126.com
Regulation of Gene Expression
Chromatin
epigenetic control
Protein degradation RNA silencing
转录水平的调控(transcriptional regulation)
常见转录因子的结构域 (domain)
TF
DNA结合域 （DNA binding domain)
Basic AA (K/R) rich, positively charged 酸性激活域 (D/E-rich) 谷氨酰胺(Q)富含域 脯氨酸(P)富含域
转录激活域
(trans-activation domain)
BICOID
四种母源影响基因 的mRNA和蛋白沿 果蝇胚胎前-后轴 分布的浓度变化图
NANOS
mRNA
protein
二、基因转录调节基本要素
（一）RNA聚合酶 (RNA Polymerase)
（二）顺式作用元件 (cis-acting elements)
（三）反式作用因子(trans-acting factors)
Binding of TFIID (TBP + 11 TAFs, 800KD) to the TATA box is the first step in initiation.
+25bp
TBP: TATA binding protein TAFs: TBP associated factors
TFIIB binds to DNA and contacts RNA polymerase near the RNA exit site and at the active center, and orients it on DNA.
Near the initiation site
A little far away
SP1 stimulates transcription in presence of TAFII110
Near
SV40 early promoter
• GC boxes bound by DNA binding protein SP1 • SP1 recruits TFIID by binding TAFII110 • Partially reconstituted complex (TBP and 3 TAFs) in addition to other GTFs, Pol II leads to high levels of transcription
RNA Pol II
Basal transcriptional apparatus (TBP, TFIIs)
CTD Pi
Many Transcriptional Activators
Factors involved in gene expression include RNA polymerase and the basal apparatus, activators that bind directly to, co-activators that bind to both activators and the basal apparatus, and regulators that act on chromatin structure (chromatin remodeling complex).
1.Transcriptional initiation 2.Histone modification 3.DNA methylation 转录后水平的调控(post-transcriptional regulation) 1. RNA silencing (siRNA and miRNA) 2. Protein degradation
CTD：RNA Pol II Cterminal domain
Phosphorylation of the CTD by the kinase activity of TFIIH may be needed to release RNA polymerase to start transcription.
What is the mechanism of activation?
?? activation) (interaction
Two models: 1.Tethering holoenzyme (recruitment) 2.Activating holoenzyme (allosteric)
In favor of recruitment model