基础分子生物学 第九讲：真核生物基因表达调控 郭红卫 hongweig@pku.edu.cn ， 新生物楼 428

1. 基础分子生物学 第九讲：真核生物基因表达调控 郭红卫 hongweig@pku.edu.cn，新生物楼428

2. 课程安排

3. 助教：冯莹 新生物楼426 dorafeng86308016@sina.com

4. 第八章 真核基因表达调控 Eukaryotic Gene Expressionand Regulation

5. 本章主要内容 • 基因表达与调控的基本概念与原理 • 转录水平的调控(transcriptional regulation): • DNA level (Genetic) • Chromatin level (Epigenetic) • 转录后水平的调控(post-transcriptional regulation)： • RNA interference （RNAi） • Protein degradation （Ubiquitin/proteasome）

6. 第 一 节基本概念与原理 Basic Concepts and Principles

7. 单个细胞 单个基因 Genome (cell’s repertoire of DNA) Transcriptome (cell’s repertoire of RNA transcripts) Proteome (cell’s repertoire of proteins) 中心法则

8. 一、基因表达的概念 • 基因组(genome) • 一个细胞或病毒所携带的全部遗传信息或整套基因。 • 基因表达(gene expression) • 基因经过转录、翻译，产生具有特异生物学功能的蛋白质分子或RNA分子的过程。 • 基因表达调控(gene regulation, or regulation of gene expression) • 基因表达是受内源及外源信号调控的。

9. Regulation of Gene Expression Chromatin epigenetic control Protein degradation RNA silencing 一般而言的基因表达调控范畴

10. 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)。 二、基因表达的时间性及空间性 （一）时间特异性

11. 人体发育过程中不同类型β-珠蛋白的含量变化

12. （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)。

13. BARD1 is expressed specifically in the apical domains ofArabidopsis inflorescence (A), ovules (B), anthers(C), and embryos (D). In suit hybridization A, B, C, D: antisense BARD1 probe; E: sense BARD1 probe as a negative control. （朱玉贤第五章课件）

14. BICOID NANOS 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 mRNA 第十章（基因和发育） protein

15. Facts • Identical genome: Virtually every cell in an organism contains a complete set of genes • Spatial specificity: But they are not all turned on in every cell or tissue • Temporal specificity: Each cell of an organism expresses a distinctive subset of genes at different time or developmental stage • Tightregulation: During development different cells express different sets of genes in a precisely regulated fashion

16. 三、基因表达的方式 按对刺激的反应性，基因表达的方式分为： （一）组成性表达 (constitutive expression) 某些基因在一个个体的几乎所有细胞中持续表达，通常被称为管家基因(housekeeping gene)。

17. Housekeeping genes • genes for essential cellular structures and metabolic pathways (e.g. rRNA, actin, tubulin) • usually expressed at high level • the level of their gene expression may vary 这类基因表达又称为组成性基因表达(constitutive gene expression)。 rRNA, actin, tubulinare commonly used as loading control in RT-PCR or Northern blot

18. （二）诱导和阻遏表达 在特定环境信号刺激下，相应的基因被激活，基因表达产物增加，这种基因称为可诱导基因 (induciblegenes)。 如果基因对环境信号应答是被抑制，这种基因是可阻遏基因 (repressible genes)。 基因表达调控大多数是对这些基因的转录和翻译速率的调节,从而导致其编码产物的水平发生改变，影响其功能。

19. 四、基因表达调控的生物学意义 （一）维持细胞增殖、分化 （二）维持个体生长、发育 （三）适应环境变化 第九、十章（基因与疾病、基因与发育）将要讲到

20. 一般而言，基因表达调控主要是发生在基因转录水平上的调节，即：mRNA合成的多少。一般而言，基因表达调控主要是发生在基因转录水平上的调节，即：mRNA合成的多少。 1. Transcripts （转录本）begin and end beyond the coding region (5’UTR and 3’UTR) transcription 2. The primary transcript is processed by: 5’ capping 3’ formation / polyA splicing 3. Mature transcripts are transported to the cytoplasm for translation

21. 五、基因转录调节基本要素 （一）RNA聚合酶 (RNA Polymerase) （二）特异DNA序列 (cis-acting elements) （三）调节蛋白 (trans-acting factors) Gene expression regulation at the level of DNA(transcriptional regulation) --highly sequence-dependent --varied regulation for different genes

22. cis-acting elements: promoters/regulatory sequences of genes trans-acting factors: proteins and RNAs that bind cis-elements and promote or repress gene expression

23. (一) RNA聚合酶 启动子、调节序列和调节蛋白通过DNA-蛋白质相互作用、蛋白质-蛋白质相互作用影响RNA聚合酶活性。 RNAPolI:rRNA, 相对活性50-70% RNAPolII: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??

24. 转录起始点 DNA En/Si Pro 编码序列 （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)。 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)

25. （三）真核基因的调节调节蛋白 反式作用因子 (trans-acting factor) 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

26. polⅡ TFⅡF TFⅡH TAF TAF TAF TFⅡA TBP TFⅡB DNA TATA 1. 基本转录因子 (general transcription factor, GTF) 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 TBP: TATA-box binding protein TAF: TBPassociated factors TFII: pol II associated TF RNA聚合酶Ⅱ在转录因子帮助下，形成的转录起始复合物

27. 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) 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。

28. 酸性激活域 (D/E-rich) 谷氨酰胺(Q)富含域 脯氨酸(P)富含域 蛋白质-蛋白质结合域 （dimerization, co-factors） 常见转录因子的结构域 (domain) DNA结合域 （DNAbindingdomain) Basic AA (K/R) rich, positively charged TF 转录激活域 (trans-activation domain)

29. 1） TF最常见的DNAbinding domain Zinc Finger bZIP Homeodomain bHLH

30. (1) 锌指(zinc finger) Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His C-terminal: α-helix binding DNA 常结合GCbox

31. (2) 碱性亮氨酸拉链 bZIP

32. (3) 碱性螺旋-环-螺旋bHLH bHLH蛋白(basic Helix-Loop-Helix)

33. 2） TF常见的trans-activation domain

34. (Activation domain is interchangeable)

35. Two-hybrid assay (protein-protein) separable functional domains Interaction Assays Design of Two-hybrid / Three-hybrid /etc… Tri-hybrid assay (protein-RNA)

36. 1.RNApolymeraseII2.promoterandenhancers3.transcriptionfactors1.RNApolymeraseII2.promoterandenhancers3.transcriptionfactors 真核基因转录起始的调控 Eukaryotic gene expression is usually controlled at the level of initiation oftranscription.

37. Ordered Assembly and Pol II Holoenzyme one-step multiple-step TFIID TFIID • Holoenzyme --- a supramolecular complex comprising Pol II, • most GTFs, and Mediator/Srb complex • In yeast, a 2MDa holoenzyme + TBP suffices for transcription

38. Sequential Assembly 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 RNApolymerase near the RNA exit site and at the active center, andorients it on DNA. Q: prok -10bp vs euk -25bp?

39. CTD：RNA Pol II C-terminal domain In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). • CTD is an unusual extension appended to the C terminus of the largest subunit of RNA polymerase II. • It comprises from 25 to 52 tandem copies of the consensus repeat heptad Y1S2P3T4S5P6S7. • S2 and S5 are major phosphorylation sites. • CTD phosphorylation cause the conversion of proline isomerization states. • Phosphorylation patterns on the CTD repeats determine different sets of associated factors, so that provide a dynamic platform to recruit different regulators of the transcription apparatus.

40. S2 & S5, the trigger for transcriptional process modulation PIC: PhosphoS5 is required for assembly of the PIC and facilitates mRNA capping via recruitment of capping enzymes. Elongation: S5 gradually becomes dephosphorylated, whereas S2 is phosphorylated. Terminating: PhosphoS2 ensures efficient 3′-RNA processing by triggering recruitment of 3′-RNA processing machinery. Ending: CTDs are free of phosphate groups; non-phosphorylated CTDs are required for RNA polymerase II to recycle and bind a promoter for the next cycle of transcription.

42. Many Transcriptional Activators i.e. CAAT GC-box Factors involved in gene expression include RNApolymerase and the basal apparatus, activators that bind directlyto, co-activators that bindto both activators and the basal apparatus, and regulators thatact on chromatin structure (chromatin remodeling complex).

43. Near the initiation site A little far away

44. 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

45. 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

46. What is the mechanism of activation? ?? (interaction activation) • Two models: • Tethering holoenzyme(recruitment) • Activating holoenzyme (allosteric)

47. In favor of recruitmentmodel （勾引模型）

48. tat protein of HIV can stimulatetranscription 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. tat tar

49. DNA-binding domain is to bring the activation domain into the vicinity of the startpoint.And activation is independent of the means of tethering. we can think of DNA-binding (or RNA-binding in the case of tat) domain as providing a "tethering" function, whose main purpose is to ensure that the activation domain is in the vicinity of the initiation complex. The notion of tethering is a more general idea that initiation requires a high concentration of transcription factors in the vicinity of the promoter. This may be achieved when activators bind to enhancers, upstream promoter elements, or in an extreme case by tethering to a newly-made RNA product.

50. 总结 • 所有激活因子的共性：识别靶位点（启动子、增强子）的特异性由DNA结合域决定。 • DNA结合域将转录激活域带到基础转录区域附近。 • 直接作用的激活因子具有DNA结合域和转录激活域。 • 没有转录激活域的激活因子可能与具有转录激活域的共激活因子一起行使功能。 • 基础转录区域中许多元件是（共）激活因子的靶位点 • RNA聚合酶可以和多种不同的转录因子相互作用，形成全酶复合物行使功能。