一

The Central Dogma

(replication)DNAà (transcription)àRNAà(translation)àProtein

Cistron（顺反子） is the genetic unit defined by the cis/trans test; equivalent to gene.

 

Exon is any segment of an interrupted gene that is represented in the mature RNA product.

Intron is a segment of DNA that is transcribed, but removed from within the transcript by splicing together the sequences (exons) on either side of it.

RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA.

Structural gene codes for any RNA or protein product other than a regulator.

Interrupted genes are expressed via a precursor RNA. Introns are removed when the exons are spliced together. The mRNA has only the sequences of the exons.

Pseudogenes are inactive but stable components of the genome derived by mutation of an ancestral active gene. Pseudo genes are dead ends of evolution

假基因（pseudogene）具有与功能基因相似的序列，但由于有许多突变以至于失去了原有的功能。

Overlapping genes occur in the relatively simple situation in which one gene is part of the other.

Transposable gene（转位基因） is a DNA sequence able to insert itself at a new location in the genome (without any sequence relationship with the target locus)

Housekeeping genes (constitutive gene) are those (theoretically) expressed in all cells because they provide basic functions needed for sustenance of all cell types.

看家基因是维持细胞最低限度功能所不可少的基因, 如编码组蛋白基因、编码核糖体蛋白基因、线粒体蛋白基因、糖酵解酶的基因等。这类基因在所有类型的细胞中都进行表达，因为这些基因的产物对于维持细胞的基本结构和代谢功能是必不可少的。

Luxury genes（奢侈基因） are those coding for specialized functions synthesized (usually) in large amounts in particular cell types

即组织特异性(tissue-specific gene)表达的基因,。这类基因与各类细胞的特殊性有直接的关系, 是在各种组织中进行不同的选择性表达的基因。如表皮的角蛋白基因、肌肉细胞的肌动蛋白基因和肌球蛋白基因、红细胞的血红蛋白基因等。

Genome is the complete set of sequences in the genetic material of an organism. It includes the sequence of each chromosome plus any DNA in organelles.

Proteome is the total number of proteins produced by an organism.

Transcriptome is the complete set of mRNAs present in a cell, tissue, or organism.

C-value: total amount of DNA in the genome (per haploid set of chromosomes).

value paradox describes the lack of relationship between the DNA content (C-value) of an organism and its coding potential.

repetitive DNA sequences:

Moderately repetitive DNA consists of relatively short sequences that are repeated typically 10-1000× in the genome.

Highly repetitive DNA consists of very short sequences (typically <100 bp) that are present many thousands of times in the genome, often organized as long tandem repeats

Neither class represents protein.

 

 

二

Nucleus: 细胞核

Nucleoid: 类核（拟核）

Nucleic acid: 核酸

Nucleoside: 核苷

Nucleotide: 核苷酸

Ribose: 核糖

Deoxyribose: 脱氧核糖

Base：碱基

 
DNA

DNA is composed of polynucleotide chains

The Two Chains of the Double Helix Have Complementary Sequences

核苷酸之间能够彼此相连形成一条糖基与磷酸脂基相间的核苷酸链。其中一个戊糖的C5通过磷酸脂基与另一个戊糖上的C3相连，如此5’-3’磷酸双脂键便形成了糖基-磷酸脂基主链，而碱基则从主链“伸出去”。

双螺旋的平均直径为2nm。沿中心轴每旋转一周有10个核苷酸。每一转的高度（即螺距）为3.4nm。

引起DNA双链构象改变有以下因素：（1）核苷酸顺序；（2）碱基组成；（3）盐的种类；（4）相对湿度。

Linking number is the number of times one strand have to be passed through the other strand in order for the two strands to be entirely separated from each other（指双螺旋DNA中两条链相互交叉的次数）

The linking number is the sum of the twist （扭转）and the writhe（缠绕）.

Twist is the number of times one strand completely wraps around the other strand（表示DNA分子一条链绕另一条链的扭转数，即DNA分子中的Watson－Crick螺旋数）

Writhe is the number of times that the long axis of the double helical DNA crosses over itself in 3-D space（即超螺旋数）

 
RNA

RNA contains ribose and uracil and is usually single-stranded

1 RNA 是一些病毒体内的遗传物质

2 作为遗传信息由DNA到蛋白质的中间传递体(mRNA)

3 作为mRNA和氨基酸之间的遗传密码子的受体(tRNA)

4 作用于RNA转录后的加工与修饰

5 参与基因表达的调控

6 一些RNAs 具有生物催化剂功能 (RNase P ribozyme, large rRNA, self-splicing introns, etc).

 
Three major classes of RNA

l         Transfer RNA (tRNA) （15％）

l         Messenger RNA (mRNA) （5％）

l         Ribosomal RNA (rRNA) （80％）

 

 

tRNA有转运RNA的作用。

tRNA一般由70－90核苷酸组成。

二级结构呈三叶草型，由氨基酸臂、二氢尿嘧啶环、反密码环、额外环和T C环五个部分组成。

氨基酸臂由7对碱基组成，富含鸟嘌呤，末端为一CCA，接受活化的氨基酸。

DHU：二氢尿嘧啶

反密码环：由7个核苷酸组成，环中部为反密码子，由3个碱基组成。

额外环：不同的tRNA具有不同大小的额外环，所以是tRNA分类的重要指标。

T C环含有假尿嘧啶核苷。

三级结构呈倒写的L型。

 

mRNA是以DNA为模板合成的。

绝大多数真核细胞的mRNA在3‘末端有一段长约200bp的polyA尾巴。

 

Prokaryotic cell

l         5S rRNA

l         16s rRNA

l         23s rRNA

 
Eukaryotic cell

l         5S rRNA

l         5.8S rRNA

l         18s rRNA

l         28s rRNA


rRNA含量大，是构成核糖体的骨架。

Ribozymes are RNA molecules that adopt complex tertiary structure and serve as biological catalysts.

核酶就是指具有催化功能的RNA。

类病毒是在高等植物中造成疾病的传染性物质，它们是很小的RNA颗粒。

 

一个典型细胞中RNA的含量约为DNA的10倍。这与RNA在细胞中行使着多种功能有关。

核酸的变性指核酸双螺旋区的氢键断裂，变成单链，并不涉及共价键的断裂

核酸变性后，在260nm处的吸收值上升，这叫增色效应(hyperchromic effect)。增色效应常可用来衡量DNA变性的程度。

A260/ A280值可以反映核酸的纯度：

Pure DNA：A260/ A280 =1.8

Pure RNA：A260/ A280 =2.0

 

Tm (melting temperature): 熔解温度，指把DNA的双螺旋结构失去一半时的温度。不同序列的DNA，Tm值不同。DNA中G－C含量越高，Tm值越高，成正比关系

 

Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources

l         Southern blot:    ss DNA  × ss DNA

l         Northern blot:    ss RNA  × ss DNA

l         Western blot:   Protein  × antibody

l         In situ hybridization  (原位分子杂交)

Probe:a labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence

Sequencing nucleic acid: Dideoxy method(双脱氧法)

Enzyme method or Sanger procedure

关键是在DNA的聚合过程中依赖特殊反应底物(2′,3′—双脱氧核苷三磷酸ddNTP)的特异性终止进行DNA测序

 

 

邻接片段（Contigs）：染色体片段的克隆，两个片段通过有重叠部分推断出两者相互邻接

 

三

Mitosis: 有丝分裂

Histone: 组蛋白

Nucleosome: 核小体

Chromosome: 染色体

Chromatin: 染色质

Chromatid: 染色单体

Centromere (中心粒)

Telomere（端粒）

Tandem gene cluster（串联基因簇）

 

Chromatin(染色质) describes the condition of the chromosomal material during the interphase (between mitoses) of the cell cycle.
Chromosome（染色体） is a discrete unit of the genome carrying many genes. Each chromosome consists of a very long molecule of duplex DNA and an approximately equal mass of proteins. It is visible as a morphological entity only during cell division.
Nucleoid（拟核）is the compact body that contains the genome in a bacterium.


Nucleosome（核小体） is the basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins.

The nucleosome is composed of a core of eight histone proteins and the DNA (core DNA, 146 bp) wrapped around them. The DNA between each nucleosome is called a linker DNA. Each eukaryote has a characteristic average linker DNA length (20-60 bp)

单体核小体大约含200bp的DNA，后者与一个组蛋白八聚体相连。每个八聚体包含H2A，H2B，H3和H4各两个，有时把这些组蛋白叫核组蛋白。核小体通过组蛋白H1相互连接，犹如一串念珠

The majority of the associated proteins are small, basic proteins called histones.

Histones are small, positively charged (basic) proteins。组蛋白具有特殊的氨基酸组成，由带有正电荷的lysine和arginine组成。

30nm的微纤丝使DNA压缩大约100倍，它是染色质的第二层次结构组织。

更高层次的组织是某些序列特异的DNA结合蛋白（一类非组蛋白）将使DNA按一定区域连接成较大型的突环，将使DNA被分成20000～100000个碱基对的突环，每一个突环含有若干功能相关的基因。

 

 
Chapter 4   DNA replication, transcription and translation
Replication

Semiconservative replication

l         DNA synthesis requires deoxynucleoside triphosphates and a primer: template junction

l         DNA is synthesized by extending the 3’ end of the primer

l         Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis

 

DNA复制的特点

l         以四种dNTP作为底物

l         反应需接受模板的指导（base-pairing）

l         链延伸需要有引物3’－羟基存在

l         链延伸方向为5’-3’

l         产物DNA的极性与模板相反

 
Replication modes

l         Replication eyes－for circular dsDNA

Replication fork is the point at which strands of parental duplex DNA are separated so that replication can proceed

l        Rolling circle replication

真核rDNA的扩增;F因子DNA的转移;λ裂解途经的复制

l         D-loop replication

高度不对称的，一条链先复制，另一条链保持单链而被取代。待一条链复制到一定程度，露出另一条链的复制起点，另一条链才开始复制。

当两条链的复制起点分开一定距离时就产生D环复制。

Replicon is a unit of the genome in which DNA is replicated. Each replicon contains an origin for initiation of replication

The origin is a sequence of DNA at which replication is initiated. 原核生物DNA分子中只有一个，真核生物有多个复制起始点

A terminus is a segment of DNA at which replication ends

 

 

 

1、oriC contains four 9 bp binding sites for the initiator protein DnaA. Synthesis of DnaA is coupled to growth rate so that initiation of replication is also coupled to growth rate.

2、DnaA forms a complex of 30-40 molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding.

3、DnaB is a helicase that use the energy of DNA hydrolysis to further melt the double-stranded DNA .

4、SSB (single-stranded binding protein) coats the unwinded DNA.

5、DNA primase load to synthesizes a short RNA primer for synthesis of the leading strand.

6、Primosome: DnaB helicase and DNA primase

 

与DNA聚合反应有关的酶包括多种DNA聚合酶和DNA连接酶。

DNA polymerase is able to synthesize DNA from four deoxynucleoside 5’-triphosphates (dNTP) as long as a DNA molecular to be copied is provided.

DNA polymerases are enzymes that synthesize a daughter strand(s) of DNA (under direction from a DNA template). May be involved in repair or replication.
DNA replicase is a DNA-synthesizing enzyme required specifically for replication.
一个能够在模版链上合成新的DNA链的酶叫做DNA聚合酶。原核和真核生物中都包含有多种DNA聚合酶的活动。

Only one DNA polymerase is the replicase. The others participate in repair of damaged DNA.

 

DNA polⅠ

l         5` → 3`聚合酶活性: DNA复制中起辅助作用

l         3` → 5`外切酶活性：校对功能

l         5` → 3`外切酶活性：引物切除、损伤修复

主要功能是切除引物，填补冈崎片段产生的空隙及DNA损伤的修复

用特异的蛋白酶处理，可把DNA polⅠ水解为两个片段:

l         小片段：323个氨基酸残基， 5` → 3`外切酶活性

l         大片段或称Klenow片段：604个氨基酸残基，DNA聚合酶活性和3` → 5`外切酶活性

 

DNA pol Ⅲ consists of multiple subunits (α、ε、θ、τ、δ、δ`、β、κ、γ、ψ)，共十个。

Holoenzyme（全酶）refers to an enzyme that contains several different subunits and retains some activity even when one or more subunits is missing

Core enzyme（核心酶）

l         α亚基： 5` → 3`聚合酶活性

l         ε亚基：3` → 5`外切酶，校对和编辑

l         θ亚基：组建核心酶复合体

pol Ⅲ是原核生物体内真正起复制作用的酶

 

DNA ligase

Ligation of Okazaki fragments are linked by DNA ligase

催化双链DNA切口处的5’磷酸基和3’羟基生产磷酸二酯键

 

DNA discontinuous replication
The leading strand of DNA is synthesized continuously in the 5’-3’ direction.

The lagging strand of DNA must grow overall in the 3’-5’ direction and is synthesized discontinuously in the form of short fragments (5’-3’) that are later connected covalently.

Okazaki fragments （冈崎片断）are the short stretches of 1000-2000 bases produced during discontinuous replication; they are later joined into a covalently intact strand.

Semidiscontinuous replication is mode in which one new strand is synthesized continuously while the other is synthesized discontinuously.


A helicase is an enzyme that uses energy provided by ATP hydrolysis to separate the strands of a nucleic acid duplex.

The single-strand binding protein (SSB) attaches to single-stranded DNA, thereby preventing the DNA from forming a duplex

 

 

 

Elongation: three different DNA polymerases are involved

1.      DNA pol α: 2. DNA pol δ: 3. DNA pol ε

 

酵母细胞：

l         自主复制序列（ARS）autonomously replicating sequences

l         起点识别复合物（ORC）origin recognition complex

 

7 Polymerase Chain Reaction (PCR)

（1）Denaturation,（2）annealing,（3）extension,（4）cycle through 25-35 times

l         DNA template,

l         A pair of oligo DNA primers （启动DNA扩增,Taq DNA聚合酶（催化DNA合成）,

l         Buffer（提供DNA合成所需pH、离子强度等环境）,

l         dNTP（DNA合成的原料）

 

 

translation

Translation machinery

（1）      mRNAs (~5% of total cellular RNA)

（2）      tRNAs (~15%)

（3）      aminoacyl-tRNA synthetases (氨酰tRNA合成酶)

（4）      ribosomes

 

Terminator: A specific DNA sequence where the transcription complex dissociate

The protein coding region of each mRNA is composed of a contiguous, on-overlapping string of codons called an opening reading frame (ORF)

Ribosome binding site (RBS) in prokaryotic mRNA, complementary with the sequence at the 3’ end of 16S rRNA

 
The ribosome is composed of a large and a small subunit

The large subunit contains the peptidyl transferase center, which is responsible for the formation of peptide bonds.

The small subunit interacting with mRNA contains the decoding center, in which charged tRNAs read or “decode” the codon units of the mRNA.

 

In cells, the small and large ribosome subunits associate with each other and the mRNA, translate it, and then dissociate after each round of translation. This sequence of association and dissociation is called the ribosome cycle.

 

Ribosomes is unable to discriminate between correctly or incorrectly charged tRNAs (是否携带正确的氨基酸)

 

 

Polysome/polyribosome: an mRNA bearing multiple ribosomes


Function site of ribosome

l         A site（或称 acceptor site）(aminoacyl-tRNA)

l         P site (或称 donor site) (peptidyl-tRNA)

l         E site (Exit site)  (deacylated-tRNA)

 

 

At the heart of protein synthesis is the translation of nucleotide sequence information into amino acids. This work is accomplished by tRNA.

tRNA are adaptors between codons and amino acids

 
Translation process

（1）      initiation-the assembly of a ribosome on an mRNA molecule.

（2）      elongation-repeated cycles of amino acid addition.

（3）      termination-the release of the new protein chain.

 

Initiation of protein synthesis in Prokaryote

l         Start codon: AUG (codes for methionine)

l         Start aa: methionine

l         Initiator  tRNA: fMet－tRNAf （N－甲酰甲硫氨酰－tRNA）

l         30S preinitiation complex

l        70S initiation complex: 30S preinitiation complex + 50S subunit

Elongation of peptide chain

Three stage：

（1）      Loading of aminoacyl-tRNA：codon-anticodon recognition

（2）      Transferation of peptide chain：formation of peptide bond

（3）      Translocation of mRNA

 

New amino acids are attached to the C-terminus of the growing polypeptide chain.

Protein is synthesized in a N- to C- terminal direction

Peptide bonds are formed by transfer of the growing peptide chain from peptidyl- tRNA to aminoacyl-tRNA.

RF:release factors

 

起始  在mRNA结合位点上的30S小亚基通过50S大亚基和氨基酰-tRNA结合而与大亚基连接

延伸  核糖体沿着mRNA移动，肽链通过从肽酰tRNA转移到氨酰tRNA而延伸。

终止  多肽链从tRNA上释放。核糖体从mRNA上解离下来

 

 
Chapter 4  Regulation of gene expression

原核生物基因表达的调控可以在DNA、转录、翻译三个不同层次进行调控，但转录水平的调控是最主要的方式，也是最经济、最有效的方式。

Housekeeping genes : expressed constitutively, essential for basic processes involving in cell replication and growth.

Inducible genes : expressed only when they are activated by inducers or cellular factors.

 

Promoters:RNA polymerase bind to and initiate transcription at sites called promoters, generally found near points at which RNA synthesis begins on the DNA template.

 

Gene Expression is Controlled by Regulatory Proteins

Positive regulators or activators enhance the RNA-promoter interaction, so INCREASE the transcription

Negative regulators or repressors impede access of RNA polymerase to the promoter, so DECREASE or ELIMINATE the transcription

 
Part 1 Regulation in prokaryotes
1 Regulation of Transcription Initiation

sequences that code for trans-acting products; and cis-acting sequences that function exclusively within the DNA. Gene activity is regulated by the specific interactions of the trans-acting products (usually proteins) with the cis-acting sequences (usually sites in DNA).

 

Operon: a unit of prokarytoic gene expression and regulation which typically includes:

（1）      Structural genes

（2）      Control elements, such as operator.

（3）      Regulator gene(s) whose products recognize the control elements.

 
The lactose (Lac) Operon

The enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source.

LacZ, lacY, lacA

Lactose/allolactose is a native inducer to release  RNA transcription from Plac.

IPTG (isopropyl-b-D-thiogalacto-pyranoside, 异丙基硫代-β-D-半乳糖苷 ), a synthetic inducer, can rapidly stimulate transcription of the lac operon structural genes. ® IPTG is used to induce the expression of the cloned gene from lac promoter in many vectors, such as pUC19.

 

An activator and a repressor together control the lac genes

The activator: CAP (Catabolite Activator Protein，分解代谢基因激活蛋白) or CRP (cAMP Receptor Protein); responses to the glucose level.

The repressor: lac repressor that is encoded by LacI gene; responses to the lactose.

 

l         When glucose is present

The level of cAMP is low in cell, and  CRP exists as a dimer which can’t  bind to DNA  to regulate transcription.

l         When glucose is absent

The level of cAMP increase and CRP bind to cAMP.The CRP-cAMP complex binds to Plac just upstream from the site for RNA polymerase. Induces a 90°bend in DNA, enhance RNA polymerase binding to the promoter and transcription by 50-fold.


协调调节(coordinate regulation)

负调节与正调节协调合作

阻遏蛋白封闭转录时，CAP不发挥作用

如没有CAP加强转录，即使阻遏蛋白从P上解聚仍无转录活性

☆葡萄糖/乳糖共同存在时，细菌优先利用葡萄糖

葡萄糖可降低cAMP浓度，阻碍其与CAP结合从而抑制转录

结论：lac操纵子强的诱导作用既需要乳糖又需缺乏葡萄糖

 

 

 

2 Regulation after Transcription Initiation

Amino acid biosynthetic operons are controlled by premature transcription termination: the tryptophan operon

The trp operon encodes five structural genes required for tryptophan synthesis.

These genes are regulated to efficiently express only when tryptophan is limiting.

Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation

The trp operon is a single transcription unit (7kb ) downstream of Ptrp and Otrp.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The Trp repressor（色氨酸阻遏物 ）
The attenuator 弱化子/衰减子

Regulation by attenuation is common for amino acid synthesis (>6 operons),
The importance of attenuation

（1）      A typical negative feed-back regulation

（2）      Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan.

（3）      Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation.

（4）      Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead.

 
Part 2  Regulation in eukaryotes
Similarity of regulation between eukaryotes and prokaryote

1.Principles are the same: signals, activators and repressors, recruitment and allostery, cooperative binding

2.Expression of a gene can be regulated at the similar steps, and the initiation of transcription is the most pervasively regulated step.

Difference in regulation between eukaryotes and prokaryote

1.Pre-mRNA splicing adds an important step for regulation.

2.The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart.

3.Nucleosomes influence access to genes.

4.Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes.

 

Transcription of a single gene may be regulated by many different factors interacting with regulatory elements upstream or downstream of the transcribed sequence.

 

- The activity of a transcription factor can be assigned to separate protein domains

l         Activation domains.

l         DNA-binding domains.

l         Dimerization domains.

l         Ligand（配体）-binding domains. ain.

 
DNA-binding domains

（1）      The helix-turn-helix domain

（2）      The zinc finger domain

（3）      Leucine zipper motif

（4）      Helix-Loop-Helix proteins

螺旋－折叠－螺旋结构基序由两段α螺旋和连接它们的β转角结构组成，一般长约22个AA残基，由其中靠近C端的一段螺旋与DNA大沟中的碱基直接接触，该螺旋称为识别α螺旋。另一段螺旋没有特异性，与DNA骨架相接触。HTH蛋白与DNA结合时可形成对称的同二聚体。

锌指结构有两种：一种是Cys2/His2类型，另一种是Cys2/Cys2锌指。

亮氨酸拉链中α螺旋的突出特点是每隔7个AA残基出现一个Leu。这种出现频率使Leu大都集中排列在α螺旋的一侧，借助Leu测链的疏水相互作用形成同向平行的拉链状，进而形成二聚体结构。

螺旋－突环－螺旋型DNA结合蛋白是近年来新发现的一种新型的DNA结合蛋白，由40～50个AA残基形成两个两性的α螺旋，中间由长约9～20个氨基酸残基间隔的可变连接区（突环），通过兼性的螺旋疏水面相互作用形成二聚体。

 

The activating regions(转录激活结构) are grouped on the basis of amino acids content

• Acidic activation domains

• Glutamine-rich domains

• Proline-rich domains

Some eukaryotic activators bind to DNA sites called enhancers that are quite distant from the promoter, influencing the rate of transcription at a promoter that maybe located thousands of base pairs away.

Specific elements called insulators（绝缘子） control the actions of activators, preventing the activating the non-specific genes

locus control region (LCR) ，GCR (global control region)

 

Gene “silencing” is a position effect－a gene is silenced because of where it is located, not in response to a specific environmental signal.

转基因植物和转基因动物中往往会遇到这样的情况，外源基因存在于生物体内，并未丢失或损伤，但该基因不表达或表达量极低，这种现象称为基因沉默（gene silence）。这是基因工程中遇到的一个影响实际应用的重要问题。一般认为基因沉默有三种情况：位置效应的基因沉默、转录水平的基因沉默和转录后水平的基因沉默。

Eukaryotic Gene Regulation at Steps After Transcription Initiation