1. Chapter 4 Transcription in Eukaryotes: Mechanism and Regulation
4.1 Eukaryotic RNA
polymerases
4.2 Eukaryotic Promoters
4.3 General Transcription
Factors and Initiation
4.4 Specific Transcription Factors
and Transcriptional Regulation
4.5 Structures of specific
transcription factors
4.6 Experiments
4.1 真核生物RNA聚合酶
4.2 真核生物启动子
4.3 通用转录因子与
转录起始
4.4 特异转录因子与
转录调控
4.5 特异转录因子的结构
4.6 实验研究
In chapter 4, we will learn about the mechanism and regulation of transcription in eukaryotes. Chapter 4 has 6 sections. They are 4.1 Eukaryotic RNA polymerases; 4.2 Eukaryotic Promoters; 4.3 General Transcription Factors and Initiation; 4.4 Specific Transcription Factors and Transcriptional Regulation; 4.5 Structures of specific transcription factors; and 5.5 Experiments.
第4章我们将要学习的是关于真核生物转录的机理与调控。第4章包括6节，它们是：4.1 真核生物RNA聚合酶；4.2 真核生物启动子；4.3 通用转录因子与转录起始；4.4 特异转录因子与转录调控；4.5 特异转录因子的结构；4.6 实验研究。

2. Transcription in Eukaryotes: Mechanism and Regulation 真核生物转录：机理与调控
Although the basic scheme of transcription is similar between eukaryotes and prokaryotes, the detailed mechanisms are quite different. Eukaryotes have many more genes and require much more complex regulation of gene expression. This increased complexity is reflected in the complexity of the eukaryotic transcriptional mechanism.
虽然真核生物和原核生物转录的基本框架是相似的，详细的机理却有很大不同。真核生物拥有更多的基因，需要对基因表达进行更复杂的调控。这种的复杂性反映在真核生物转录机理的复杂性上。
Prokaryote
Eukaryote

3. Transcription in prokaryotes
Transcription in prokaryotes is quite simple. Only RNA polymerase holoenzyme is necessary to initiate the transcription. But eukaryotes have three different RNA polymerases.
原核生物的转录较为简单，只需要RNA聚合酶的全酶就能启动转录，而真核生物有三种不同的RNA聚合酶。

4. Transcription in eukaryotes
Specific transcription factor
General transcription factors
General transcription factors
In addition, eukaryotic transcription needs the help of proteins called transcription factors. Transcription factors can be divided into two major categories: general transcription factors and specific transcription factors. General transcription factors are required for transcription of all genes. Specific transcription factors are required for controlling transcription of specific genes.
此外，真核生物的转录还需要称为转录因子的蛋白质的帮助。转录因子可被分为两大类：通用转录因子和特异转录因子。通用转录因子是所有基因转录都需要的。特异转录因子是那些能提高或降低特殊基因转录速率的蛋白质。
Eukaryotic RNA polymerase II
and transcription factors

5. 4.1 Eukaryotic RNA polymerases 真核生物RNA聚合酶
OK, section 4.1, about eukaryotic RNA polymerases. It has been found that eukaryotes use three different RNA polymerases. Each of them transcribes different classes of genes. The three polymerases are termed RNA polymerase I, II and III. The genes they transcribed are called Class I, II, and III genes, respectively.
第4节，关于真核生物的RNA聚合酶。已经发现，真核生物具有三种不同的RNA聚合酶，每一种聚合酶转录不同类型的基因。这三种聚合酶称为RNA聚合酶I、II和III。它们转录的基因也分别称为I类、II类和III类基因。

6. Locations of RNA polymerases
Nucleolus ( RNA polymerase I )
Different polymerases are located at different places. RNA polymerase I exists in the nucleolus. RNA polymerases II and III exist in the nucleoplasm.
不同的RNA聚合酶存在于细胞核里的不同位置，其中RNA聚合酶I在核仁中，RNA聚合酶II和III在核质中。
Nucleoplasm ( RNA polymerase II and III )

7. RNA polymerase I RNA polymerase I rRNA precursor Processing 5.8S rRNA
RNA polymerase I is used to transcribe only one gene, the large rRNA precursor. This large transcript is later used to produce three mature rRNAs. That is the 5.8S rRNA, the 18S rRNA and the 28S rRNA. There are many copies of the large rRNA precursor gene in each cell. So, although RNA polymerase I only transcribes one kind of gene, it is quite active.
RNA聚合酶I只用来转录一种基因——大的rRNA前体基因。这一大的转录物之后用来产生三种成熟的rRNA（即： 5.8S rRNA、18S rRNA和28S rRNA ）。在每个细胞中有许多这一大的rRNA前体基因的拷贝。因此，虽然rRNA聚合酶I只用来转录一种基因，它却是相当活跃的。
5.8S
rRNA
18S
rRNA
28S
rRNA

8. RNA polymerase III RNA polymerase III RNA precursor Processing 5S U6
RNA polymerase III mainly transcribes the tRNA genes. It also produces some other small RNAs, including the 5s rRNA and the U6 RNA.
RNA聚合酶III也转录相对来说较少的基因，它主要转录tRNA基因。第III类基因也包含一些小的RNA基因，如5S rRNA和U6 RNA基因。 5S
rRNA U6
snRNA
4.5S
tRNA

9. RNA polymerase II RNA polymerase II RNA precursor mRNA Processing
Translation
RNA polymerase II transcribes all genes that produce mRNA. It also transcribes small RNAs called snRNAs.
RNA聚合酶II是多产的RNA聚合酶。它转录所有产生mRNA并因此编码了蛋白质的基因。它也转录一些小RNA基因，称之为snRNA。
snRNAs
Protein

10. RNA polymerase II Holoenzyme: Core enzyme (β’, β & α) + Subunit σ
RNA pol II: Core subunits (1, 2 & 3) + Subunits 4-12
RNA polymerase II is composed of 12 subunits. Rpb1, Rpb2, and Rpb3 are called the ‘core subunits’ because they are fundamentally required for enzyme activity. These subunits are related to the three subunits of the E. coli RNA polymerase core: β, β’ and α.
RNA聚合酶II由12个亚基组成。Rpb1、Rpb2和 Rpb3称为‘核心亚基’，因为它们是产生酶活性的最基本要求。这几种亚基与大肠杆菌RNA聚合酶核心的β、β’和α这三个亚基有一定的联系。

11. RNA polymerase II
The overall structure of the 12-subunit polymerase resembles a crab claw. A double-stranded DNA molecule can fit into the claw. The clamp then closes during transcription to stabilize the association between DNA and the newly polymerized RNA.
由12个亚基组成的聚合酶的总体结构也象蟹钳。钳内的空间可以容纳一条双链DNA分子。在转录中钳关闭来稳定DNA和新合成的RNA之间的联合。

12. 4.2 Eukaryotic Promoters 真核生物启动子
Prokaryotic
promoter
E. coli promoters usually have two elements, a −10 box and a −35 box. Eukaryotic promoters are much more complex. The core promoter may have four elements: the TATA box, the initiator, the TFIIB recognition element, and the downstream promoter element. Eukaryotic promoters may also have an upstream promoter elements, such as GC box and the CCAAT box.
大肠杆菌启动子通常含有两个元件，即−10框和−35框。真核生物启动子要复杂得多，其核心启动子可以包含四个元件：TATA框、起始子、TFIIB识别元件和下游启动子元件。在核心启动子以外，上游启动元件可能含有一些不同的保守序列，如GC框和CCAAT框。
Eukaryotic promoter

13. Eukaryotic Promoters 真核生物启动子
“I would like to have TATA box and Initiator as my promoter. What would you like?”
“Well, I prefer BRE and DPE.”
But a eukaryotic promoter almost never includes all of the elements just listed. Different promoters contain different combinations of these elements. For example, the promoter of one bird’s gene may have TATA box and initiator. But that of a dog’s gene may have BRE and DPE.
但是一个真核启动子几乎从来不会包括所有上面列出的元件。不同的启动子含有这些元件的不同组合。例如，在鸟类中的一个基因的启动子可能包含TATA框和起始子，而狗中某基因的启动子却具有BRE和DPE两种元件。

14. TATA box TATAAA -10 box TATAAT
The TATA box is common promoter element. It has a consensus sequence of TATAAA. Please note that this sequence is quite similar to the −10 box sequence in prokaryotes (TATAAT). Please don’t confuse these two elements. The TATA box only refers to an element in eukaryotes. The TATA box is usually centered at position −25 relative to the transcription start site, not −10.
TATA框是特别常见并且研究得很清楚的真核启动子元件。它的名称来源于它的共有序列，即在非模板链上的TATAAA。注意这一序列与原核生物的−10框序列（TATAAT）非常相似。然而，这二个元件不应该被混淆。TATA框只是指真核生物中的一个元件，它通常以转录起始位点上游−25位为中心，而不是−10位。

15. Specialized genes have TATA boxes
Housekeeping genes –
Genes of development –
TATA box is often present in specialized genes that make proteins only found in particular cells. Housekeeping genes, and genes that are important for organismal development generally do not have TATA boxes.
TATA框最常出现在特殊的基因里，这些基因只在特殊的细胞里产生蛋白。在持家基因和一些对生物的发育起重要作用的基因中没有TATA框。

16. DPE: Downstream Promoter Element
G CG
The downstream promoter element (DPE) is another common promoter element in eukaryotes. It is located downstream of the transcription start site, at position +30, approximately. The consensus sequence of the DPE is G(A/T)CG, meaning that the consensus sequence may have A or T at the second position.
下游启动子元件（DPE）是另一个很常见的启动子元件。它开始于转录起始位点下游约+30的位置。DPE的共有序列是G(A/T)CG，意思是共有序列在第二个位置可以是A或T。

17. Initiator / 起始子 T PyPyAN PyPy A
The consensus sequence of the initiator (Inr) promoter element is is PyPyAN(T/A)PyPy. ‘Py’ refers to Pyrimidine – T or C. ‘N’ stands for any nucleotide. This sequence is positioned with A at the transcription start site. The initiator itself can drive basic transcription, but its function is greatly amplified by the presence of other promoter elements.
起始子（Inr）启动子元件的共有序列是PyPyAN(T/A)PyPy。‘Py’指嘧啶，即T或C。‘N’代表任何核苷酸。这个序列的A被放在转录起始位点上。起始子本身能够驱动基础转录，但在其它启动子元件存在的情况下它的功能会变得更强。

18. BRE: TFIIB Recognition Element
GGG
CCA
CGCC
The TFIIB recognition element (BRE) is located upstream of the TATA box. It has the consensus sequence (G/C)(G/C)(G/A)CGCC. As the name suggest, the BRE sequence binds to TFIIB. TFIIB is a general transcription factor. We will discuss it afterwards.
TFIIB识别元件（BRE）位于TATA框的上游，具有共有序列(G/C)(G/C)(G/A)CGCC。正如这一名称所暗示的，BRE序列结合到TFIIB上，TFIIB是通用转录因子中的一种，我们将在后面讨论。

19. 4.3 General Transcription Factors and Initiation（通用转录因子与转录起始）
Eukaryotic RNA
polymerase II:
“I need help from
other proteins.”
Prokaryotic RNA
polymerase
So far, we have learned that eukaryotes have three kinds of RNA polymerases and the promoter structure of class II genes is very complex. What is more, eukaryotic RNA polymerases cannot bind to promoters by themselves. They need help from other proteins. These proteins are the general transcription factors, TFIIA, B, D, E, F, and H. General transcription factors are required for transcription of all class II genes. The collection of general transcription factors with RNA polymerase II at the promoter is called the pre-initiation complex.
到现在为止，我们已经学习了，真核生物具有三中RNA聚合酶，并且II类基因启动子的结构是非常复杂的。更有甚者，真核生物RNA聚合酶光靠自身还不能结合到启动子上。它们需要其它蛋白质的帮助，这些蛋白质是通用转录因子，即TFIIA、 B、 D、 E、 F和H。通用转录因子在转录所有II类基因中都需要。通用转录因子与RNA聚合酶II在启动子位置的组合称为前起始复合体。
pre-initiation complex

20. TFIID
The first TFII to bind the promoter is TFIID. It is a large protein. It is composed of 8-10 subunits. One of these subunits is called TBP, the TATA-binding protein. TBP is found in most eukaryotes and its amino acid sequence is quite conserved.
第一个结合到启动子上的是TFIID。这是一个由8-10个亚基组成的大蛋白。这些亚基中的一个是称为TATA结合蛋白（TBP）的蛋白质，它与TATA框结合。TBP在大多数真核生物中都有，它的氨基酸序列在非常不同的生物中也相当保守。

21. TBP: TATA-Binding Protein
The shape of TBP is like a saddle. In the ‘stirrups’ of the protein are phenylalanine residues. The phenylalanine R group contains an aromatic ring that resembles a nitrogenous base in size and shape. When TBP binds to the minor groove of the DNA, this ring can insert between the DNA bases and cause it to bend sharply. The bend leads to the separation of the DNA double strands so that a single-stranded template can be exposed for transcription. The weak binding between T-A base pairs allows the TATA box to be denatured relatively easily.
TBP的形状类似于马鞍。在蛋白质的‘马镫’处是苯丙氨酸残基。苯丙氨酸的R基团含有一个芳香环，它在大小和形状上类似与含氮的碱基。当TBP结合到DNA的小沟上时，这个环可以插入到DNA的碱基之间并引起DNA链的明显弯曲，形成一个将近80º的角。这种弯曲使DNA双链很容易分开，从而暴露出单链模版用于转录。T-A碱基对之间弱的结合力使TATA框相对较为容易发生变性。

22. Show 3D structure of TBP File: TBP-TATA box complex 1.val
Here is a picture showing the binding of TBP with TATA box.
这是一张显示TBP与TATA框结合的图片。
File: TBP-TATA box complex 1.val

23. TAFIIs: TBP-Associated Factors II
The other subunits of TFIID are called TAFIIs, the TBP-Associated Factors II.
TFIID的其它亚基称为TBP相关因子II（TAFII）。

24. TAFIIs: 1) Binding to DPE or Inr
TAFIIs have two major functions in transcription. First, they bind to DPE or Initiator, but not the TATA box, to stimulate transcription.
TAFIIs在转录中具有两种主要功能。第一，它们结合到核心启动子元件（如DPE和Inr）上而不是结合到TATA框上去促进转录。

25. TAFIIs: 2) Binding to Activator
Second, they bind to specific transcription factors to allow enhancement or inhibition of transcription.
第二，它们与特异转录因子结合以增强或抑制转录。

26. Other TFIIs
Other general transcription factors also play very important roles in forming the pre-initiation complex. As we have said, TFIID is the first protein that binds to the promoter. Actually, it needs help from TFIIA. After that, TFIIB joins them, followed by TFIIF and RNA polymerase II. TFIIE and TFIIH bind last. Since it is such a complex process, we are not going to give further details here.
其它通用转录因子在形成前起始复合体中也具有重要作用。如我们已经说过的那样，TFIID是第一个结合到启动子上的蛋白质。实际上，它需要TFIIA的帮助。之后，TFIIB加入进来，随后是TFIIF和RNA聚合酶II。TFIIE和TFIIH最后结合上来。因为这一过程是如此复杂，在此我们不对它进行详细的讨论。

27. Now showing Initiation of Transcription
We have a short movie to watch now.
现在我们来看一个短片。
File: biophoto7 \ Life \ Initiation of transcription (5’)

28. 4.3.3 General Transcription factors for RNA Polymerase I and III RNA聚合酶 I 和III的通用转录因子
Eukaryotic RNA polymerases I and III also have their own general transcription factors. UBF and SL1 are the two general transcription factors for RNA polymerase I. TFIIIB and TFIIIC are the two for RNA polymerase III.
真核生物RNA聚合酶I和III也具有它们自身的通用转录因子。UBF与SL1是RNA聚合酶I的通用转录因子，TFIIIB与TFIIIC是RNA聚合酶III的两个通用转录因子。

29. 1. General TFs of RNA polymerase I
Transcription initiation for class I genes is much simpler than for class II genes. Only two general transcription factors are required. They are UBF and SL1. The Class I promoter contains two elements, a core element centered at the transcription start site and an upstream promoter element. SL1 binds to the core element and helps recruit RNA polymerase I to the promoter. UBF binds to the upstream promoter element to help SL1 to bind to the core element.
I类基因的转录起始要比II类基因的简单得多。它只需要两个通用转录因子，即UBF和 SL1。I类启动子含有两个元件，即一个以转录起始位点为中心的核心元件和一个上游启动子元件。SL1结合到核心元件上并帮助召集RNA聚合酶I到启动子位置。UBF结合到上游启动子元件帮助SL1结合到核心元件上。

30. 2. General TFs of RNA polymerase III
Transcription initiation of Class III genes is more varied. For tRNA genes, the promoter contains two elements, box A and box B, both of which are downstream of the transcription start site. A class III general transcription factor called TFIIIC binds to these elements. TFIIIC helps another protein, TFIIIB to bind to the DNA near the transcription start site.
III类基因的转录起始有更多不同之处。对tRNA基因来说，启动子含有两个元件，即A框和B框，两者都位于转录起始位点的下游。一个称为TFIIIC的III类通用转录因子结合到这些元件上。TFIIIC帮助另一种蛋白质，即TFIIIB结合到靠近转录起始位点的DNA上。

31. General TFs of RNA polymerase III
TFIIIB helps RNA Polymerase III to bind at the gene. Even though TFIIIC detaches after transcription initiation, TFIIIB stays bound and can be used for several rounds of transcription.
TFIIIB又帮助RNA聚合酶III结合到该基因上。即使在转录起始后TFIIIC已经从DNA上脱离，TFIIIB仍然与DNA结合并在后面几轮转录中加以利用。

32. 4.4 Specific Transcription Factors and Transcriptional Regulation 特异转录因子与转录调控
Now, let’s move onto section 4.4, about Specific Transcription Factors. We need to know that the general transcription factors and RNA polymerase II rarely join together by themselves. When there are only these proteins present, transcription occurs at a low rate. This is called basal transcription. In order to change the rate of transcription, eukaryotic cells need the help from specific transcription factors.
现在，我们要转到第4节了，关于特异转录因子。要知道，通用转录因子和RNA聚合酶II很少自发地结合到一起去启动转录。当只有这些蛋白质出现的时候，转录以很低的速率进行，这称为基础转录。为了改变转录的速率，真核生物采用另一类称为特异转录因子的蛋白质。

33. Specific Transcription Factors and Transcriptional Regulation 特异转录因子与转录调控
Activators / 激活蛋白
Repressors / 阻遏蛋白
There are two types of specific transcription factors. They are activators and repressors.
特异转录因子分为两种，它们是激活蛋白和阻遏蛋白。

34. Activators / 激活蛋白
Specific transcription factors that enhance the rate of transcription are called activators.
能提高转录速率的特异转录因子称为激活蛋白。
Help recruit the pre-initiation complex.
Help organize the subunits of the pre-initiation complex, and maintain cohesion of the complex.
Attract enzymes to loosen DNA from proteins, freeing important regions like the promoter.
First, let’s look at the activators. Specific transcription factors that enhance the rate of transcription are called activators. These proteins work through a variety of mechanisms. First, they can help recruit the pre-initiation complex. Second, they can help organize the subunits of the pre-initiation complex, and maintain cohesion of the complex. And thirdly, they can attract enzymes to loosen DNA from proteins, freeing important regions like the promoter.
我们先来看激活蛋白。能提高转录速率的特异转录因子称为激活蛋白。这些蛋白通过多种不同的机理发挥作用。第一，激活蛋白能帮助召集前起始复合体；第二，它们也能帮助组织前起始复合体的亚基并保持复合体的结合力；第三，它们能够吸引酶来从蛋白上松开DNA，让启动子这样的重要区域游离出来。

35. 1. Help recruit the pre-initiation complex
Let’s first have a look at the example of an activator that helps recruit the pre-initiation complex. Here, an activator binds to DNA, attracts the general transcription factor TFIID. Then TFIID attracts other general transcription factors (including TFIIA, B, E, F, and H) and RNA polymerase II to form the pre-initiation complex.
我们先来看激活蛋白帮助召集前起始复合体的例子。在这儿呢，激活蛋白结合到DNA上，吸引通用转录因子TFIID，之后TFIID吸引其它通用转录因子（包括TFIIA, B, E, F, and H）以及DNA聚合酶II形成前起始复合体。

36. 2. Help organize the subunits of the pre-initiation complex
Secondly, activators can help organize the subunits of the pre-initiation complex and maintain cohesion of the complex. Here, the activator holds the general transcription factors IIB and IIF together. This can maintain the stability of the pre-initiation complex.
第二，激活蛋白能帮助组织前起始复合体的亚基并保持复合体的结合力。这儿呢，激活蛋白将通用转录因子IIB与IIF保持在一起。这种作用能起到稳定前起始复合体的作用。

37. 3. Attract enzymes to loosen DNA
Thirdly, activators can attract enzymes to loosen DNA from proteins and set the promoter free. Here, an activator recruits enzyme that loosens DNA from nucleosomes. This makes the promoter accessible to the general transcription factors and RNA polymerase.
第三，激活蛋白能够吸引酶来从蛋白上松开DNA，让启动子这样的重要区域游离出来。 这儿呢，激活蛋白将能使DNA从核小体上松开的酶召集起来，将DNA松开，就有可能将启动子区域游离出来以便启动基因的转录。

38. Repressors / 阻遏蛋白
Specific transcription factors that prevent the transcription of a gene are called repressors.
能阻碍基因转录的特异转录因子称为阻遏蛋白。
Blocking the activity of activators.
Blocking access of the pre-initiation complex to the promoter.
Recruiting enzymes that tighten DNA around proteins.
Now, let’s look at what functions repressors can have. Specific transcription factors that prevent the transcription of a gene are called repressors. These proteins also work through a variety of mechanisms. First, the repressors can function by blocking the activity of activators. Secondly, they can function by blocking access of the pre-initiation complex to the promoter. And thirdly, they can function by recruiting enzymes that tighten DNA around proteins, making the promoter and other important regions not available for transcription.
我们再来阻遏蛋白具有哪些作用。能阻碍基因转录的特异转录因子称为阻遏蛋白。这些蛋白也通过多种不同的机理发挥作用。第一，阻遏蛋白能通过阻碍激活蛋白的活性而发挥作用；第二，阻遏蛋白能通过阻止前起始复合体接近启动子而发挥作用，第三，阻遏蛋白能通过召集酶使DNA紧密地缠绕在蛋白上，让启动子和其它重要区域不可接近，从而阻止转录。

39. 1. Blocking the activity of activators
Firstly, repressors can function by blocking the activity of activators. Because transcription occurs very slowly when there are no activators, this alone can cause strong repression of a gene.
首先，阻遏蛋白能通过阻碍激活蛋白的活性而发挥作用。因为在缺少激活蛋白的情形下转录发生得很慢，单是这样就已经能对基因实施强的阻遏作用。

40. 2. Blocking access of the pre-initiation complex to the promoter.
Secondly, repressors can function by blocking access of the pre-initiation complex to the promoter. Here, TFIID cannot bind to the promoter. No pre-initiation complex can form at the promoter site.
第二种情况，阻遏蛋白能通过阻止前起始复合体接近启动子而发挥作用。这里，TFIID不能结合到启动子上，在启动子位点不能形成前起始复合体。

41. 3. Recruiting enzymes that tighten DNA around proteins
And thirdly, repressors can function by recruiting enzymes that tighten DNA around proteins. Here, a repressor attracts an enzyme that tightens DNA around histones. As a result, the promoter region is packed on histones tightly. It is therefore not available for transcription.
第三，阻遏蛋白能召集酶使DNA紧密地缠绕在蛋白上。这里，阻遏蛋白能够吸引酶来使DNA紧密地缠绕在组蛋白上；结果，启动子区域与组蛋白紧紧地包在一起，没法用来转录。

42. 4.4.3 Enhancers and Silencers 增强子和沉默子
Specific transcription factors rely on DNA sequences to tell them how each gene should be regulated. These sequences are called enhancers or silencers, depending whether they bind activators or repressors, respectively.
特异转录因子依靠DNA序列来告诉它们应该怎样调控每一种基因。这些序列称为增强子或沉默子，分别依赖于它们结合的是激活蛋白还是阻遏蛋白。
Specific transcription factors rely on DNA sequences to tell them how each gene should be regulated. These sequences are called enhancers or silencers, depending whether they bind activators or repressors, respectively.
特异转录因子依靠DNA序列来告诉它们应该怎样调控每一种基因。这些序列称为增强子或沉默子，分别依赖于它们结合的是激活蛋白还是阻遏蛋白。

43. Enhancers and Silencers
Transcription
is activated
Activator
Enhancer
Transcription
is repressed
This means that, if a piece of DNA is used to bind activators, it will be called an enhancer. And if a piece of DNA is used to bind repressors, it is called a silencer.
这儿的意思是：如果一段DNA用于结合激活蛋白，就将它称为增强子；如果一段DNA用于结合阻遏蛋白，就将它称为沉默子。
Repressor
Silencer

44. DNA looping / DNA成环
Enhancers and silencers are sometimes located very far from the rest of the gene. In this case, DNA is looped around, so that these regions can come quite near to the promoter.
增强子和沉默子有时位于离基因几千个碱基对远的位置。这种情况下，DNA会弯过来，这样这些距离远的调控区域就可以来到启动子的附近。

45. Sea Urchin Endo 16 gene A red sea urchin 红海胆
The regulatory regions of eukaryotic genes can become extremely complex. For example, the Endo 16 gene in Sea Urchins contains six regulatory enhancers and silencers upstream of the promoter which can respond to over a dozen different proteins. Complex regulation is necessary in this instance because Endo 16 is a gene that guides the development of sea urchin embryos.
真核基因的调控区域可以变得极其复杂。例如海胆中的Endo 16基因在启动子上游含有六个起调控作用的增强子和沉默子，它们能够响应十几种不同蛋白的调控作用。在这个例子中复杂调控是必须的，因为Endo 16是指导海胆胚胎发育的基因。
A red sea urchin
红海胆

46. 4.5 Structures of Specific Transcription Factors 4.5 特异转录因子的结构?
Eukaryotic
Now, let’s look at section 4.5, about Structures of Specific Transcription Factors. We have learned that, in both prokaryotes and eukaryotes, the binding of proteins to DNA is required for transcription and its regulation. But how can these proteins bind to DNA? In this section, we will discuss the motifs of these proteins that make these proteins bind tightly with their target DNA.
现在，我们来看一看第5节，关于特异转录因子的结构。我们已经学习过，在原核生物和真核生物中转录和转录调控都需要有蛋白质与DNA发生结合。那么，蛋白质是怎么能实现这样的结合的呢？本节我们就来学习一些能与DNA结合的蛋白质结构基序。
Prokaryotic ?

47. Major Groove vs. Minor Groove
A ≠ T
G≠ C
Minor groove
A ≈ T
G ≈ C
First, we need to know that the major grooves and the minor grooves of the DNA double helix look very different. A, G, T, and C look different only from the major groove. From the minor groove, G looks like C, and A looks like T. Therefore, most DNA-binding proteins recognize specific DNA sequences at the major groove.
首先，我们应该知道，DNA的大沟和小沟看起来很不一样。A、G、T和C只能从DNA大沟的表面才能完全加以区别。从小沟上看，G与C没有区别，A与T没有区别。因此，绝大多数识别特异序列的DNA结合蛋白都结合到DNA的大沟上。

48. 4.5.1 DNA-Binding Motifs in Prokaryotes 原核生物DNA结合基序
The helix-turn-helix
(HTH) motif
Let’s first look at DNA-Binding Motifs in Prokaryotes. Most prokaryotic DNA-binding proteins have the helix-turn-helix (HTH) motif. The HTH motif is made of two α helices connected by a short peptide turn. Here, helix 1 is the recognition helix. It fits tightly into the major groove of DNA.
我们先来看原核生物DNA结合基序。大多数原核生物DNA结合蛋白都含有螺旋-转角-螺旋（HTH）基序HTH基序由通过一个短的肽链转角连接起来的两个α螺旋组成。在此，螺旋1是识别螺旋，它的大小正好适合紧密地插入到大沟中。
Recognition
helix

49. Negative Regulation – the lac Repressor 负调控──lac阻遏蛋白
The lac repressor is one example of a prokaryotic DNA-binding protein with HTH motif.
Lac阻遏蛋白是原核生物DNA结合蛋白具有HTH基序的一个例子。

50. Show 3D structure of the lac repressor
The HTH motifs can be clearly seen in the 3D structure of a lac repressors binding to its target DNA.
其HTH基序在它与目标DNA结合的3D结构图中可以清楚地看出来。
File: lac repressor with DNA.val

51. The trp repressor
The trp repressor also has the HTH motif. It can only bind to its DNA when there is tryptophan.
trp阻遏蛋白也具有HTH基序，它只有在存在色氨酸的情况下才能结合到DNA上。

52. The trp repressor DNA Trp E. coli Trp repressor Operator
The trp repressor is actually a dimer of two proteins. Each protein has its own HTH motif. Tryptophan is clearly seen in this picture.
trp阻遏蛋白实际上是两个蛋白的二聚体，两个蛋白各含有一个HTH基序。色氨基酸在这张图片里清晰可见。
Trp repressor

53. Show 3D structure of the trp repressor
Let’s take a look at the 3D structure of trp repressor dimer binding to its target DNA.
我们来看一看trp阻遏蛋白二聚体结合到目标DNA上的3D结构图。
File: trp repressor.val

54. 4.5.2 DNA-Binding Motifs in eukaryote 真核生物DNA结合基序
1. The homeodomain
同源异型域
2. Zinc finger motif
锌指基序
3. Leucine zipper
亮氨酸拉链
HLH motif
螺旋-环-螺旋基序
Eukaryotic
Prokaryotes mainly have one HTH motif we have just discussed. Eukaryotes have many more kinds of DNA binding motifs. The typical DNA binding motifs in eukaryotes include 1) The homeodomain, 2) Zinc finger motif, 3) Leucine zipper, and 4) Helix-loop-helix motif.
原核生物主要有HTH这一种基序，真核生物中有更多种类的DNA结合基序。典型的真核生物DNA结合基序有四种：1）同源异型域，2）锌指基序，3）亮氨酸拉链，4）螺旋-环-螺旋基序。 ?

55. 1. The homeodomain / 同源异型域
Similar to HTH
of prokaryotes
The homeodomain is a common motif in proteins involved in organismal development. The homeodomain contains three α helices connected by short peptide turns. Here, helix 1 and helix 2 together appear quite similar to the HTH motif of prokaryotes. Helix 1 makes specific contacts in the major groove. Helix 2 provides support. And helix 3 has a peptide extension that fits into the minor groove of the DNA.
同源异型域是与生物发育有关的蛋白质中一种常见的基序。同源异型域包含由短的肽链转角连接在一起的三个α螺旋。其中的两个螺旋在一起看起来与HTH基序非常相似，其中一个起支撑作用，另一个与DNA大沟发生特异性接触。同源异形域的第三个螺旋具有一个肽链延伸段，它正好适合深入到DNA的小沟中。

56. Mutation of homeotic genes
Homeodomains are found in proteins coded by homeotic genes. Homeotic genes are responsible for developing the correct organs at correct positions. If there is a mutation in the homeotic gene, there may be very strange transformation of body parts. For example, in fruit fly, a mutation called antennapedia causes legs to grow on the head, where antennae [-teni:] would normally be.
同源异形域是在由同源异形基因编码的蛋白质中发现的。同源异形基因负责在正确的位置发育出正确的器官，如果同源异形基因发生突变，就有可能出现生物身体部件的畸形。例如，在果蝇中，一种称为触角足畸形的突变导致了足长到了头部上本来应该长触角的地方。

57. Show 3D structure of homeodomain
3D structure of homeodomain binding to its target DNA has also been available.
同源异形域与目标DNA结合的3D图也已得到。
File: Homeodomain.val
Baird-Titus JM, et al. The solution structure of the native K50 Bicoid homeodomain bound to the consensus TAATCC DNA-binding site J. Mol. Biol. v356, p

58. 2. Zinc finger motif / 锌指基序
The second DNA-binding motif in eukaryotes is zinc fingers. In this motif, an α helix and an anti-parallel β strand are held together by a zinc ion. The structure is elongated and looks like a finger. That is why it is called zinc finger. The finger shape can easily insert into the major groove. So, the protein can bind to the DNA tightly.
真核生物中第二类DNA结合基序是锌指。在这一基序中，一个α螺旋和一条反向平行的β链被一个锌离子保持在一起。这个结构会伸展成有点像手指那样的形状。这种手指形状可以容易地插入到DNA大沟中，使蛋白与DNA紧密地结合在一起。

59. Show 3D structure of zinc fingers
File: Zinc finger.val
Finger 1
Finger 2
One protein often has several zinc finger motifs together. This is very helpful to increase the stability of DNA-protein complex. The 3D structure here has three zinc fingers together.
DNA结合域常常具有几个互相靠近的锌指基序，这非常有利于增强DNA-蛋白质复合体的稳定性。图片显示3个锌纸结构连在一起的情况。
Finger 3

60. 3. Leucine zipper / 亮氨酸拉链 Leucines
The third DNA-binding motif we are going to talk is the leucine zipper. The leucine zipper is composed of two α helices of two proteins. Each protein has a line of leucines. Leucines have hydrophobic R groups. It is the hydrophobic interaction of these R groups that brings the two helices together. The helices coil slightly around each other, perhaps giving the impression of a zipper.
我们要讲的第三种DNA结合基序是亮氨酸拉链。亮氨酸拉链由两种蛋白质上的两个α螺旋组成，每种蛋白质具有一排亮氨酸。亮氨酸带有疏水性R基团，正是由于疏水相互作用使两个螺旋紧紧地靠在一起。两个螺旋互相靠近并发生轻微卷曲，或许正是这给我们留下了拉链的印像。

61. Heterodimerization / 异源二聚化作用
Leucine zippers can also form between two different proteins. This is called heterodimerization. The heterodimers can recognize different DNA sequences than the leucine zipper made from only one kind of protein. This property can be used to create more DNA-binding domains from fewer parts.
两种不同的蛋白质组合在一起也能形成亮氨酸拉链，这叫做异源二聚化作用。与从只有一种蛋白质产生的亮氨酸拉链相比，异源二聚体亮氨酸拉链能够识别不同的DNA序列。这一特性能够使较少种类的蛋白产生出更多种类的DNA结合域。

62. Show 3D structure of leucine zipper
File: Leucine zipper.val
A 3D structure of leucine zipper is also available.
亮氨酸拉链的3D结构图也已得到。
Yeast cells
Wu SW, et al. Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates Proteins v60, p

63. 4. Helix-loop-helix motif / 螺旋-环-螺旋基序
The helix-loop-helix
(HLH) motif
The last DNA-binding motif is the helix-loop-helix. It is quite similar to the leucine zipper. Like the leucine zipper, it is also composed of two separate proteins. HLH can also heterodimerize. Unlike the leucine zipper, however, the dimerization is realized through HLH motif. The HLH motif is composed of two α helices connected by a loop.
最后一种DNA结合基序是螺旋-环-螺旋基序，它与亮氨酸拉链很相似。像亮氨酸拉链一样，HLH是由两个蛋白质形成的，它也像一把钳子一样抓住DNA。HLH也可以发生异源二聚化。然而，与亮氨酸拉链不同，二聚化是通过HLH基序实现的。HLH基序由通过一个环连接在一起的两个α螺旋组成。

64. HLH versus HTH The helix-loop-helix (HLH) motif The helix-turn-helix
(HTH) motif
The helix-loop-helix should not be confused with the helix-turn-helix. HLH is one of the DNA-binding motifs found in eukaryotes. And HTH is the main DNA-binding motif of prokaryotes.
注意不要将螺旋-环-螺旋结构与螺旋-转角-螺旋相混淆。 HLH基序是真核生物DNA结合基序中的一种，而HTH基序是原核生物中的主要DNA结合基序。

65. Show 3D structure of HLH File: HLH.val
Let’s have a look at the 3D structure of mouse HLH.
我们来看一看小鼠HLH基序的3D结构。
Ferre-D'Amare AR, et al. Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain Nature v363, p.38-45

66. 4.6 Experiments / 实验研究 5.5.1 RNA polymerase targets RNA聚合酶的目标
Modularity of Specific
Transcription Factors 特异转录因子的模块化
OK, the last section we are going to discuss is about the experiments made to find out the RNA polymerase targets and the modularity of Specific Transcription Factors.
呃，我们要讨论的最后一节关于两项实验研究方面的内容。一是关于RNA聚合酶的目标，二是关于特异转录因子的模块化。

67. 4.6.1 RNA polymerase targets RNA聚合酶的目标
First, about RNA polymerase targets. We have already learned that eukaryotes have three different RNA polymerases. We also learned that RNA polymerase I mainly produces rRNA. RNA polymerase II mainly produces mRNA and RNA polymerase III mainly produces tRNA. But, how did we know?
首先，关于RNA聚合酶的目标。我们已经知道，真核生物拥有三种不同的RNA聚合酶，其中RNA聚合酶I主要产生rRNA，RNA聚合酶II主要产生mRNA，RNA聚合酶III主要产生tRNA。但是，一开始是怎么知道的呢？

68. α- amanitin / α鹅膏蕈碱
Actually, the activity of the different polymerases was determined using α-amanitin. Alpha-amanitin is produced by a kind of mushroom and is deadly poisonous to human beings.
实际上，不同聚合酶的活性是使用了一种叫做α鹅膏蕈碱的毒素来测定的，这种毒素是一种蘑菇产生的，对人的毒性非常大。

69. Why is α- amanitin poisonous?
Why is alpha-amanitin so poisonous to human beings? Studies indicated that alpha-amanitin inhibits the three eukaryotic RNA polymerases to very different extents. Low concentrations completely inhibit RNA polymerase II, but have no effect on the activity of the polymerases I and III. Much higher concentrations can also inhibit RNA polymerase III, but still have no effect on RNA polymerase I.
为什么α鹅膏蕈碱对人类有这么大的毒性？研究发现，该毒素对三种不同的真核RNA聚合酶的抑制程度有很大差别。低浓度的毒素完成抑制RNA聚合酶II，而对其它聚合酶的活性没有任何影响。更高浓度的毒素也能抑制RNA聚合酶III，但仍然对RNA聚合酶I没有影响。

70. Functions of RNA polymerases
High concentration
of α-amanitin
RNA polymerase I
RNA polymerase II
RNA polymerase III
rRNA precursor
In order to see which genes are transcribed by which polymerase, cells are exposed to α-amanitin. It was found that, under very high concentrations of α-amanitin, only rRNA precursor is produced. This means that rRNA precursor is produced by RNA polymerase I.
为了弄清哪些基因由哪种聚合酶转录，细胞被暴露在α鹅膏蕈碱中。暴露在很高浓度α鹅膏蕈碱的细胞只产生rRNA前体，意味着这正是RNA聚合酶I所转录的基因。
RNA polymerase I
produces rRNA
precursors.

71. Functions of RNA polymerases
Low concentration
of α-amanitin
RNA polymerase I
RNA polymerase II
RNA polymerase III
rRNA precursor
tRNA, 5S rRNA …
Then, cells expose to low concentrations of the toxin also produce tRNA and other small RNAs like the 5S rRNA, suggesting that these are the genes transcribed RNA polymerase III. mRNA is not produced even at low concentrations of α-amantin, suggesting that RNA polymerase II is responsible for transcription of mRNA.
而暴露在低浓度毒素中的细胞还能产生tRNA和其它小RNA如5S rRNA，意味着这些基因是由RNA聚合酶III转录的。即使在低浓度α鹅膏蕈碱下mRNA也不能产生，说明RNA聚合酶II负责转录mRNA。
RNA polymerase III
produces tRNA, 5S rRNA and other snRNAs.

72. 4.6.2 Modularity of Specific Transcription Factors 特异转录因子的模块化
Module
exchange
The second experiment we are going to look at is the one to find out the modularity of specific transcription factors. Modularity means that a protein is composed of separate modules. For example, a specific transcription factor is composed of one DNA binding module and one transcription activation module. When two such proteins exchange their modules, they will still be able to function.
我们要看的第二个实验研究是关于怎么发现特异转录因子模块化的。模块化的意思是一种蛋白质由不同的模块组成，例如，特异转录因子含有一个DNA结合模块和一个分开的激活模块，如果两种蛋白交换它们的模块，交换后两者仍然具有功能。

73. The reporter genes / 报告基因
physicalsciences.ucsd.edu/outreach/image_gall...
RFP
GFP
Before explaining the experiments to demonstrate the modularity of specific transcription factors, let’s first understand what a reporter gene is. A reporter gene is a gene whose transcriptional and/or translational products can be easily detected and measured. For example, genes coding for GFP and RFP proteins are very frequently used as reporter genes. GFP stands for green fluorescent protein. This protein can emit green fluorescent light under visible light.
在解释特异转录因子的模块化实验研究内容之前，我们先来了解一下什么是报告基因。报告基因就是转录和/或翻译产物可以很容易地被检测出来的基因。例如，编码GFP和RFP蛋白的基因常用作报告基因。GFP就是绿色荧光蛋白，它在可见光下能发出绿色荧光。
Brain cancer models  Human U-87-RFP glioma growing in brain of nude mouse.
脑瘤模型：生长在裸鼠脑中的人神经胶质瘤

74. The modularity of Gal4
Modularity is very obvious in the activator Gal4. Gal4 binds to a sequence called UAS (upstream activation sequence) to activate transcription. In the experiment, first, a UAS sequence was placed in front a reporter gene. Thus, it is convenient to see if the activator protein works. It was seen that when the UAS was present in front of the reporter gene, GAL4 activated transcription of the gene.
模块化最明显的例子是Gal4。Gal4结合到一段称为UAS的序列去激活转录。首先，把一段UAS序列放到一个报告基因的前面。报告基因是一个转录情况可以容易地被探测和测定的基因。这样，就能方便地试验一种激活蛋白工作情况的好坏。当UAS出现在报告基因前面的时候，GAL4激活了这个基因的转录。

75. The modularity of Gal4
Next, the DNA-binding module of GAL4 protein was replaced with that of LexA. Now, transcription is not activated, because the hybrid activator cannot bind to UAS.
其次，用一种经过人工改造过的蛋白替换GAL4蛋白。这样，转录不能被激活，因为产生的Gal4-LexA蛋白不能结合到UAS上。

76. The modularity of Gal4
Finally, a LexA-operator sequence was placed in front of a reporter gene. Now, transcription is activated, because the Gal4-LexA protein was able to bind to the DNA. This shows what modularity means. In this experiment, as long as the activation domain of Gal4 could be brought near the target gene, its function was not affected by the identity of the DNA-binding domain.
最后，一段LexA-操纵基因序列被放到报告基因的前面。现在，因为这种Gal4-LexA蛋白能够结合到DNA上，转录被激活了。这显示了模块化的含义。本实验中，只要Gal4的激活域能够被吸引到靠近目标基因的位置，它的功能就不会因为DNA结合域的不同而受影响。