分子生物学(杨洋)第七章 翻译(2)
[分享]分子生物学名词解释1
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分子生物学名词解释第二章(主要的:核小体、半保留复制、复制子、单链结合蛋白、岗崎片段、错配修复、DNA的转座、C值矛盾、前导链与后随链。
)1. C值反常现象(C值矛盾C-value paradox):C值是一种生物的单倍体基因组DNA的总量。
真核细胞基因组的最大特点是它含有大量的重复序列，而且功能DNA序列大多被不编码蛋白质的非功能DNA所隔开，这就是著名的“C值反常现象”。
C值一般随着生物进化而增加，高等生物的C值一般大于低等生物。
某些两栖动物的C值甚至比哺乳动物还大，而在两栖动物里面，C值变化也很大。
2.DNA的半保留复制:由亲代DNA生成子代DNA时，每个新形成的子代DNA中，一条链来自亲代DNA，而另一条链则是新合成的，这种复制方式称半保留复制。
3.DNA聚合酶:●以DNA为模板的DNA合成酶●以四种脱氧核苷酸三磷酸为底物●反应需要有模板的指导●反应需要有3 -OH存在●DNA链的合成方向为5 34.DNA连接酶（1967年发现）：若双链DNA中一条链有切口，一端是3’-OH，另一端是5‘-磷酸基，连接酶可催化这两端形成磷酸二酯键，而使切口连接。
但是它不能将两条游离的DNA单链连接起来DNA连接酶在DNA复制、损伤修复、重组等过程中起重要作用5.DNA 拓扑异构酶(DNA Topisomerase)：拓扑异构酶І：使DNA一条链发生断裂和再连接，作用是松解负超螺旋。
主要集中在活性转录区，同转录有关。
例：大肠杆菌中的ε蛋白拓扑异构酶Π：该酶能暂时性地切断和重新连接双链DNA，作用是将负超螺旋引入DNA分子。
同复制有关。
例：大肠杆菌中的DNA旋转酶6. DNA 解螺旋酶/解链酶（DNA helicase)通过水解ATP获得能量来解开双链DNA。
E.coli中的rep蛋白就是解螺旋酶，还有解螺旋酶I、II、III。
rep蛋白沿3 ’ 5’移动，而解螺旋酶I、II、III沿5 ’ 3’移动。
7. 单链结合蛋白(SSBP-single-strand binding protein)：稳定已被解开的DNA单链，阻止复性和保护单链不被核酸酶降解。
分子生物学：第七章翻译1
• 1965年Khorana利用已知的核苷酸重复序列，以此多聚核苷酸作模 板，在体外进行蛋白质合成。
1、组成重复的RNA只能是UCU-CUC,翻译的多肽为丝氨酸、亮氨 酸排列，此处不能确定其密码子
2、怎么读都产生多聚苯、多聚丝、多聚亮。1与2只有一个密码 子相同，都有丝氨酸、亮氨酸，不能确定
3、怎么读都为四个密码子的循环，但合成的肽连中氨基酸有三 种。亮氨酸、苏氨酸、酪氨酸 3与2对照，彼此共有密码子CUU和亮氨酸，因此，亮氨酸码 子为CUU 2中已知CUU是亮氨酸，2与1对照丝氨酸一定为UCU，因为1、 2中都有丝氨酸。苯丙氨酸则为UUC
• 1961年Crick 和Brenner证明遗传密码是三联体密码子。
• 原黄素(二氨基吖啶)可以使DNA增加或减少一个碱基。
(-)(-)(-)
一次删去5’-GUA ↓ ↓ ↓ UAC GGA U………3’
读码框不变
1961年Nirenberg 的无细胞蛋白合成系统：
（1）去模板：用DNAase处理E.coli抽提物，使DNA降解，除去原有的细菌 模板。这样的抽提物中含有：
• TψC常由5bp的茎和7nt和环组成。此臂负责和核糖体上的rRNA 识别 结合；
• 反密码子臂(anticodon arm)常由5bp的茎区和7nt的环区组成，它负 责对密码子的识别与配对。
• D环 (D arm)的茎区长度常为4bp，含有二氢尿嘧啶（dihydrouracil）。 负责和氨基酰tRNA聚合酶结合;
二人获得1968年诺贝尔化学奖
遗传密码特点：
• 连续性：密码子之间是连续的，中间没有停顿。如果插入或删除一 个碱基将会发生错误，称为移码，由移码产生的突变，称为移码突 变。
• 不重叠性：一般情况下，密码子之间是相连的，之间没有重叠 （non-overlapping）。但有例外。密码子的重叠与重叠基因是不同 的概念。
分子生物学7第七章 蛋白质翻译ppt课件
d、按paracodon在tRNA上的位置(氨基 酸序列) 将AARS分为两类
type I 包括 Val,Arg,Gln,Glu,Ile,Leu,Met,Trp,Tyr paracodon 大多位于反密码子臂
type II paracodon 大多位于氨基酸接受臂 个别还同时在附加臂上有相应碱基
二、 mRNA
★ 蛋白质翻译是基因表达的第二步 ★ tRNA在翻译过程中起“译员”的作用 ★ 参与翻译的RNA 除tRNA外，还有rRNA和mRNA
★ tRNA 既是密码子的受体,也是氨基酸的受体
★ tRNA 接受AA要通过氨酰tRNA合成酶及其自身的 paracodon 的作用才能实现
★ tRNA 通过其自身的 anticodon而识别 codon ★ 密码子有自身的特性
c、 paracodon 的特征
--- 同一种AARS 所识别的一组同功受体具有相同的 副密码子
tRNA
Ala (GGC)
tRNA
Ala (UGC)
具有G3 ：U70 paracodon
--- paracodon 是为AARS（特定氨基酸）所识别的若干 碱基（并非均为一对核苷酸），且位置不固定。
--- 尽管副密码子不是单独与AA发生相互作用，但是副密码 子可能与AA的侧链基团有某种相应性。
三联体 前两个重要 摇摆性 通用性 有一定的使用频率
★ 多种翻译因子组成翻译起始复合物，完成翻译的起始、 延伸和终止，并且保证其准确性
第一节 基本元件
一、 tRNA
最小的 RNA，4S，70 ~ 80个NT 1、 tRNA的高级结构
1964 Holly. R. 鉴定出 tRNAphe 的二级结构为三叶草 形（77个NT）
分子生物学名词解释
分子生物学名词解释1.翻译(translation)：以mRNA为模板，氨酰-tRNA为原料直接供体，在多种蛋白质因子和酶的参与下，在核糖体上将mRNA分子上的核苷酸顺序表达为有特定氨基酸顺序的蛋白质的过程。
2.密码子(codon)：mRNA中碱基顺序与蛋白质中氨基酸顺序的对应关系是通过密码实现的， mRNA中每三个相邻的碱基决定一个氨基酸，这三个相邻的碱基称为一个密码子。
3.密码的简并性(degeneracy)：—个氨基酸具有两个以上密码子的现象。
4.同义密码子(synonym codon)：为同—种氨基酸编码的各个密码子，称为同义密码了。
5.变偶假说(wobble hypothesis)：指反密码子的前两个碱基(3’-端)按照标准与密码子的前两个碱基(5’-端)配对，而反密码子中的第三个碱基那么有某种程度的变动，使其有可能与几种不同的碱基配对。
6.移码突变(frame-shift mutation)：在mRNA中，假设插入或删去一个核苷酸，就会使读码发错误，称为移码，由于移码而造成的突变、称移码突变。
7.同功受体(isoacceptor)：转运同一种氨基酸的几种tRNA称为同功受体。
8.反密码子(anticodon)：指tRNA反密码子环中的三个核苷酸的序列，在蛋白质合成过程中通过碱基配对，识别并结合到mRNA的特殊密码上。
9．多核糖体(polysome)：mRNA同时与假设干个核糖体结合形成的念珠状结构，称为多核糖体。
1．中心法那么(central dogma)：生物体遗传信息流动途径。
最初由Crick(1958)提出，经后人的不断补充和修改，现包括反转录和RNA复制等内容。
2．半保存复制(简称复制)〔semiconservative replication)：亲代双链DNA 以每条链为模板，按碱基配对原那么各合成一条互补链，这样一条亲代DNA双螺旋，形成两条完全相同的子代DNA螺旋，子代DNA分子中都有一条合成的“新〞链和一条来自亲代的旧链，称为半保存复制。
分子生物学名词解释
分子生物学名词解释分子生物学名词解释1. 基因（顺反子）（gene(cistron)）：指能产生一条肽链的DNA 片段。
包括编码区和其上下游区域（引导区和尾部），以及在编码片段间（外显子）的割裂序列（内含子）。
2. DNA聚合酶（DNA polymerase）：合成子代DNA链（在DNA模板的指导下）的酶。
任何独特的酶可在修复或复制（或两者都有）中发挥作用。
3. RNA聚合酶（RNA polymerase）：使用DNA作为模板合成RNA的酶（正式应为DNA依赖性RNA聚合酶）。
4. 反转录酶（reverse transcriptase）：以单链RNA为模板合成双链DNA的酶。
5. A deoxyribonuclease(DNAase)is an enzyme that attacks bonds in DNA. It may cut onlyone strand or both strand.DNA酶：攻击DNA之间化学键的酶。
（第二句自译：它可能仅仅切断单链或双链。
）6. RNA酶（ribonucleases(RNAase)）：底物为RNA的酶，它可对双链或单链RNA特异性作用，它可为核酸内切酶或核酸外切酶。
7. 核酸外切酶（exonuclease）：每次可从核酸链一头切割一个核苷酸的酶，可能特异性切割DNA或者RNA的5‘或者3’端。
8. 核酸内切酶（endonuclease）：切割核酸链内的化学键。
可特异性地切割RNA或者单链或双链DNA。
9. A hotspot is a site in the genome at which the frequencyof mutation (or recombination)is very much increased, usually by at least an order of magnitude relative to neighboring sites.热点：突变或重组频率显著增加的位点。
分子生物学单词翻译
分子生物学单词翻译由生物技术08级共4个班全体同学翻译整理者叶德佶(1).2'-deoxyribose2'-脱氧核糖A five-carbon sugar that differs from ribose in having a hydrogen instead of a hydroxyl group at the 2'position. The sugar is a distinctive component of DNA, whose backbone is an alternating copolymer of 2'deoxyribose and phosphate.与核糖不同，在2号位由羟基取代氢原子的五碳糖。
它是DNA主要识别部分，DNA随着2号位五碳糖和磷酸基团的改变而使其变化。
(2).3' splice site3末端剪接位点A sequence overlapping the junction at the 3' end of an intron and the 5' end of the downstream exon. The sequence is required for proper splicing of that intron and used to be called the "acceptor" site.3末端内含子和5末端下游外显子交界处重叠序列，它要求内含子上“受体”位点的正确剪接。
(3).3' untranslated trailer region (3' UTR)3末端非编码区拖尾A noncoding sequence located downstream (3') of the coding region in an mRNA. The 3' UTR sometimes contains recognition sequences for the binding of cytoskeletal proteins such as tubulin, which can localize the mRNA within specific regions of the cell.在mRNA下游编码区的非编码序列。
分子生物学(杨洋)课程总结-2014
Nucleosomes are the building blocks of chromosomes（核小体是 染色体的结构单位）
在真核细胞中大多数DNA被包装进核小体
The nucleosome is composed of a core of eight histone proteins and the DNA (core DNA, 147 bp，核心DNA) wrapped around them（核小体由8个组蛋白所形成的核组成， DNA缠绕在组蛋白核上）
Molecular Biology of Gene
（基因的分子生物学）
Gene concept Gene structure Gene replication Gene expression Gene recombination Gene mutation
第二章-染色体，染色质和核小体 (Chromosomes, chromatin, and the nucleosome）
• •
• 复制中产生的错误以及它们是如何被修复的
• 自发产生或来自外界的攻击造成的各种损伤 • 细胞修复损伤的多种修复机制
生物在保持遗传物质忠实性方面的努力
• 复制中产生的错误以及它们是如何被修复的 • 自发产生或来自外界的攻击造成的各种损伤—DNA损伤的各种类型 • 细胞修复DNA损伤的多种修复机制： – 1. Direct reversal of DNA damage by photoreactivation (光活化作 用) and alkyltransferase (烷基转移酶)-直接将损伤逆转 – 2. 剪切修复系统（excision repair system)： • 仅仅将受损的核苷酸去除 • 将包含损伤的一小段单链DNA去除 – 3. 重组修复系统（Recombination repair）：当DNA两条链都受损 （断裂）时采用这种修复—双链断裂修复（ Double-strand break repair） – 4. 移损DNA合成（Translesion DNA synthesis）：DNA聚合酶的复 制进程被受损碱基阻碍时采用的修复
(完整版)分子生物学》试题及答案
《分子生物学》考试试题B课程号：66000360 考试方式：闭卷考试时间：一、名词解释（共10题，每题2分，共20分）1. SD 序列2. 重叠基因3．ρ因子4．hnRNA5. 冈崎片段、6. 复制叉(replication fork)7. 反密码子(anticodon)：8. 同功tRNA9. 模板链(template strand)10. 抑癌基因二、填空题（共20空，每空1分，共20分）1.原核基因启动子上游有三个短的保守序列,它们分别为____和__区.2.复合转座子有三个主要的结构域分别为______、______、________。
3.原核生物的核糖体由_____小亚基和_____大亚基组成，真核生物核糖糖体由_____小亚基和_______大亚基组成。
4.生物界共有___个密码子，其中__ 个为氨基酸编码，起始密码子为__ _______；终止密码子为_______、__________、____________。
5. DNA生物合成的方向是_______，冈奇片段合成方向是_______。
6.在细菌细胞中，独立于染色体之外的遗传因子叫_______。
它是一种_______状双链DNA，在基因工程中，它做为_______。
三．判断题（共5题，每题2分，共10分）1.原核生物DNA的合成是单点起始，真核生物为多点起始。
( )2.在DNA生物合成中，半保留复制与半不连续复制指相同概念。
( )3.大肠杆菌核糖体大亚基必须在小亚基存在时才能与mRNA结合。
( )4.密码子在mRNA上的阅读方向为5’→ 3’。
( )5.DNA复制时，前导链的合成方向为5’→ 3’，后随链的合成方向也是5’→ 3’。
（）四、简答题（共6题，每题5分，共30分）1.简述三种RNA在蛋白质生物合成中的作用。
2.蛋白质合成后的加工修饰有哪些内容?3.简述人类基因组计划的主要任务。
4.简述现代分子生物学的四大研究热点。
分子生物学名词解释
1.翻译(translation)：以mRNA为模板，氨酰-tRNA为原料直接供体，在多种蛋白质因子和酶的参与下，在核糖体上将mRNA分子上的核苷酸顺序表达为有特定氨基酸顺序的蛋白质的过程。
2.密码子(codon)：mRNA中碱基顺序与蛋白质中氨基酸顺序的对应关系是通过密码实现的，mRNA中每三个相邻的碱基决定一个氨基酸，这三个相邻的碱基称为一个密码子。
3.密码的简并性(degeneracy)：—个氨基酸具有两个以上密码子的现象。
4.同义密码子(synonym codon)：为同—种氨基酸编码的各个密码子，称为同义密码了。
5.变偶假说(wobble hypothesis)：指反密码子的前两个碱基(3’-端)按照标准与密码子的前两个碱基(5’-端)配对，而反密码子中的第三个碱基则有某种程度的变动，使其有可能与几种不同的碱基配对。
6.移码突变(frame-shift mutation)：在mRNA中，若插入或删去一个核苷酸，就会使读码发错误，称为移码，由于移码而造成的突变、称移码突变。
7.同功受体(isoacceptor)：转运同一种氨基酸的几种tRNA称为同功受体。
8.反密码子(anticodon)：指tRNA反密码子环中的三个核苷酸的序列，在蛋白质合成过程中通过碱基配对，识别并结合到mRNA的特殊密码上。
9．多核糖体(polysome)：mRNA同时与若干个核糖体结合形成的念珠状结构，称为多核糖体。
1．中心法则(central dogma)：生物体遗传信息流动途径。
最初由Crick(1958)提出，经后人的不断补充和修改，现包括反转录和RNA复制等内容。
2．半保留复制(简称复制)（semiconservative replication)：亲代双链DNA以每条链为模板，按碱基配对原则各合成一条互补链，这样一条亲代DNA双螺旋，形成两条完全相同的子代DNA螺旋，子代DNA分子中都有一条合成的“新”链和一条来自亲代的旧链，称为半保留复制。
分子生物学 翻译
fMet fMet
Tu GTP
5'
AUG
3'
进 位
成肽
转 位
（四）真核生物延长过程
真核生物肽链合成的延长过程与原核 基本相似，但有不同的反应体系和延长因 子。
另外，真核细胞核蛋白体没有E位，转 位时卸载的tRNA直接从P位脱落。
三、肽链合成的终止
当mRNA上终止密码出现后，多肽链 合成停止，肽链从肽酰-tRNA中释出， mRNA、核蛋白体等分离，这些过程称 为肽链合成终止。
胞浆 胞浆
tRNA rRNA
74-95个核苷酸
28S，5400个核苷酸 18S，2100个核苷酸 5.8S，160个核苷酸 5S， 120个核苷酸
转运氨基酸 与密码子识别
构成核糖体 ， 蛋白质合成场 所
S：沉降系数 （1S=10-13秒）
碱基数量：bp, Kb, Mb
原核生物16S rRNA的二级结构
（一）原核生物翻译起始复合物形成
• 核蛋白体大小亚基分离； • mRNA在小亚基定位结合； • 起始氨基酰-tRNA的结合； • 核蛋白体大亚基结合。
1. 核蛋白体大小亚基分离
IF-1 IF-3
2. mRNA在小亚基定位结合
5'
AUG
3'
IF-1
IF-3
S-D序列：
在原核生物mRNA起始密码AUG上 游，存在4~9个富含嘌呤碱的一致性序列， 如-AGGAGG-，称为S-D序列。又称为核 蛋白体结合位点（ribosomal binding site，RBS)
氨基酸的活化形式：氨基酰－tRNA 氨基酸的活化部位：α－羧基 氨基酸与tRNA连接方式：酯键 氨基酸活化耗能：2个~P
分子生物学翻译
2 Materials and methods2.1 Cell culture and antibodyHL-60 leukemic cells were grown in suspension in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 0.5% antibiotics and 2.0 g/L sodium bicarbonate in a 5% CO2 humidified incubator at 37 °C. The monoclonal antibody (MAb) to nucleophosmin/B23 (37/5.1) was kindly provided by Dr. P.K. Chan, Department of Pharmacology, Baylor College of Medicine, Houston, Texas. Characterization of nucleophosmin/B23 MAb has been reported previously [12].2.2.OligonucleotidesThe phosphorothioate analogs of deoxyoligonucleotides corresponding to nucleotides —2 to 18 of the c-myc cDNA were synthesized in both of the reverse (5’-GCT ACG GGG AGT TGC AAT CG-3’)and antisense (5'.GCT AAC GTT GAG GGG CAT CG-3’) orientations (ASIA Company Ltd., Oregon-Wilsonville).2.3. Induction of differentiationFor the induction of differentiation, HL-60 cells (2 x 105 cells/ml) were incubated with 1 or 5μMRA (all-trans). The percentages ofCD-11b-positive cells and fluorescence intensity were evaluated by FACScan (FACScan, Beckton-Dickinson) using specific anti-CD11bFITC-conjugated MAb (Sigma).2.4. Western blottingSeparated proteins on SDS-PAGE were electrotransferred to Hy-bond-PVDF membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). The PVDF membrane was then soaked in a blocking solution [5%(w/v) non-fat milk in TBST buffer (20 mM Tris-HCI. pH 7.5, 0.5 M NaCl, and 0.1% (v/v) Tween 20)]for 1 h at room temperature. The soaked PVDF membrane was then incubated with MAb against nucleophosmin/B23 (diluted 1:2000 in 3% (w/v) non-fat milk in TBST) for 1 h at room temperature, washed with TBST buffer three times for 15min each and incubated at room temperature for 1 h in horseradish peroxidase conjugated goat anti-mouse IgG antibody (diluted 1:2000 in TBST buffer). The membrane was washed three times with TBST for 15 min each. Immunobands were identified by en hanced chemiluminescence reaction (ECL, Amersham Pharmacia Biotech).2.5 Transfection with antisense c-Myc oligonucleotidesHL-60 cells were seeded at a density of 3 × 105 per well in 1.0 ml OPTI-MEM I reduced serum medium (Gibco). One to 5 micromolar oligonucleotide (c-Myc reverse or antisens e) and 6 μg lipofectin reagents in OPTI-MEM I reduced serum medium were mixed gently. The mixture was incubated for 45 min at room temperature, added to the HL-60 cellsincubator.and was incubated for 24 h at 37 °C in the CO22.6 PlasmidThe 5’region of nucleophosmin/B23 gene (−552/+2217) containing c-Myc binding site was cloned into luciferase reporter gene vector pGL3 vector. Genome nucleophosmin/B23 was amplified by PCR using 5’-CGA GGT ACC TGA ACT TTG GGG TAA-3’ and 5’-TAG TCC ATG GGC CTT TAG TTC ACA ACC-3’ as primers. Amplified PCR products were then separated and isolated from 1% agarose gel. The 2.7 kb nucleophosmin/B23 genome region was then subcloned into the cloning site of the vector pGL3 supplied in the Eukaryotic TA cloning kit. The orientation of the cDNA in pGL3 was determined by nucleotide sequencing using the sequence kit (Amersham Pharmacia Biotech).2.7 Transient transfection by electroporation5 × 107cells were washed twice with ice-cold PBS, resuspended in 0.3 ml PBS and then transferre d to electrochamber containing 40 μg of reporter plasmid. Electroporation was performed using the GIBCO-BRL gene pulser with a capacitance setting of 1180 μF and a voltage setting of 260 V. After electroporation, cells were incubated on ice for 10 min andtransferred to 35 ml fresh RPMI 1640 medium containing 10% FBS. After 24 h, cells were then treated with RA for various times.2 材料和方法2.1细胞培养和抗体在37℃的5％CO 2加湿培养箱中，将HL-60白血病细胞悬浮于补充有10％热灭活胎牛血清，0.5％抗生素和2.0g / L碳酸氢钠的RPMI-1640培养基中。
分子生物学(杨洋)第七章 翻译-1
Why?
10
Outline
2020/4/4
Topic 1-4: Four components of translation machinery
mRNA
tRNA
attachment of amino acids to tRNA (aminoacyl-tRNA synthetases)
This part concerned with one of the greatest challenges in understanding the gene－ how the gene is expressed
3
2020/4/4
Ch 12: Mechanisms of transcription
mRNA：5-AUGAGUAACGCG-3 translation
Protein： Met-Ser-Asn-Ala
9
2020/4/4
Translation extremely costs
In rapid growing bacterial cells, protein synthesis consumes
因
组
的
保
基
持
RNA processing
因 组
的
Translation 表 达
6
2020/4/4
What is translation?
--it is the story about decoding the genetic information contained in messenger RNA (mRNA) into proteins
分子生物学第七章 翻译-3
Model: poly(U)-ribosome-poly(Phe) (14C-labeled) UUU--Phe poly(U)—poly(Phe)
放射性标记(14C-labeled)
Release of peptidyl-puromycin from ribosome
Detection of the 14C-labeled released peptidyl-puromycin can reflect the peptidyl transferase activity
50S subunit of ribosome alone can have peptidyl transferase activity
[14C]
[14C]
How can we distinguish the released 14C-poly(Phe)-puro from the 14C-poly(Phe)-ribosome?
We should establish a system which can detect peptidyl transferase
activity easily and in vitro(体外)
Puromycin can be used to detect the peptidyl transferase reaction in vitro
First question:
Which part of ribosome is responsible for peptidyl transferase reaction? (50S subunit or 30S subunit?)
Localization of peptidyl transferase
分子生物学(第3版中译版)知识点
分子生物学(第3版中译版)知识点Molecular biology is a fascinating and rapidly advancing field that has revolutionized our understanding of life at the most fundamental level. The third edition of the translated version of the textbook on molecular biology provides a comprehensive overview of the key concepts, principles, and techniques in this exciting discipline. From the structure and function of DNA, RNA, and proteins to the mechanisms of gene expression, regulation, and genetic engineering, the book covers a wide range of topics that are essential for understanding the molecular basis of life.One of the most important knowledge points in molecular biology is the structure and function of DNA, the molecule that carries the genetic information of an organism. The book delves into the double helix structure of DNA, the complementary base pairing that allows for faithful replication, and the central dogma of molecular biology, which describes the flow of genetic information from DNA toRNA to protein. Understanding the intricate molecular machinery involved in DNA replication, transcription, and translation is crucial for grasping the fundamental processes that drive the development, growth, andfunctioning of living organisms.Another key area of focus in molecular biology is the regulation of gene expression, which determines when and where specific genes are turned on or off in response to internal and external signals. The book explores the mechanisms of transcriptional and post-transcriptional regulation, including the role of transcription factors, enhancers, and silencers in controlling gene expression. It also discusses the epigenetic modifications that can alter the accessibility of genes to the transcriptional machinery, leading to heritable changes in gene expression without altering the underlying DNA sequence.In addition to the fundamental principles of molecular biology, the book also covers the latest advances in the field, including the applications of recombinant DNA technology and gene editing tools such as CRISPR-Cas9.These revolutionary techniques have opened up new possibilities for manipulating the genetic material of organisms, leading to the development of genetically modified organisms (GMOs), gene therapy for genetic diseases, and the editing of the human germline. Theethical and societal implications of these technologies are also discussed, highlighting the need for careful consideration of the potential risks and benefits of manipulating the genetic makeup of living organisms.Overall, the translated edition of the molecular biology textbook provides a comprehensive and up-to-date overview of the key concepts and techniques in the field. It is an invaluable resource for students, researchers, and anyone interested in gaining a deeper understanding of the molecular basis of life. The book not only covers the foundational knowledge of molecular biology but also explores the cutting-edge developments and ethical considerations that are shaping the future of the field. By delving into the intricate molecular mechanisms that underlie life processes, the book inspires a sense ofwonder and appreciation for the complexity and beauty of the biological world.。
分子生物学(二)
分子生物学（二）引言概述：分子生物学是研究生物分子结构和功能的学科。
本文将继续讨论分子生物学的相关内容，重点关注五个大点，包括蛋白质合成、基因表达调控、DNA复制、基因突变和分子诊断技术。
正文：一、蛋白质合成1. 转录和翻译的关系：RNA聚合酶合成mRNA，然后在核糖体中翻译成蛋白质。
2. 编码和非编码RNA：编码RNA包括mRNA和tRNA，而非编码RNA则不直接编码蛋白质，如rRNA和miRNA。
3. 编码RNA修饰：例如，剪接和RNA编辑，可以改变RNA序列，并对蛋白质产生重要影响。
4. 信使RNA降解：通过RNA酶的作用，mRNA可以被降解，控制蛋白质的合成量和速率。
5. 蛋白质翻译后修饰：包括磷酸化、糖基化和乙酰化等多种修饰形式，影响蛋白质的功能和稳定性。
二、基因表达调控1. 转录调控：转录因子的结合可以激活或抑制基因的转录过程，影响蛋白质的合成。
2. 染色质结构：染色质的组织结构和修饰可以影响基因的可及性，进而调控基因表达。
3. miRNA的调控作用：miRNA可以与mRNA结合，抑制其翻译或诱导降解，进而调控基因表达。
4. DNA甲基化：DNA甲基化是一种在基因调控中重要的表观遗传修饰方式，参与基因的静默。
5. 细胞信号转导：细胞内外的信号转导通路可以调控基因表达，对细胞发育和功能起重要作用。
三、DNA复制1. DNA复制的步骤：包括解旋、合成互补链和连接等多个步骤，确保DNA的准确复制。
2. DNA聚合酶：DNA聚合酶是复制DNA的主要酶类，具有高度专一性和准确性。
3. 复制起始位点选择：复制起始位点的选择是复制过程的关键步骤，受到复制起始蛋白的调控。
4. DNA损伤修复：复制过程中，可能会发生DNA损伤，细胞会通过修复机制保护DNA的完整性。
5. 复制过程的调控：多种蛋白质和调控机制参与DNA复制的调节，确保复制的顺序和精确性。
四、基因突变1. 突变的类型：包括点突变、缺失、插入和倒位等多种突变类型，影响DNA序列的改变。
分子生物学名词解释英文
1.DNA Denaturation(变性) When duplex DNA molecules are subjected to conditions of pH ,temperature,or ionic strength that disrupt base-paring interactions, the DNA molecule has lost its’native conformation, and double helix DNA is separated to single strand DNA as individual randome coils.That is, the DNA is denatured.2.Renaturation（复性）Removing the denaturation factors slowly or in proper conditions, the denaturedDNA (ssDNA) restore native structure (dsDNA) and functions. This process is dependent on both DNA concentration and time.3.Hybridization （核酸分子杂交）when heterogeneous DNA or RNA are put together, they will become toheteroduplex via the base-pairing rules during renaturation if they are complementary in parts (not completely). This is called molecular hybridization.4.Hyperchromic effect （增色效应）The absorbance at 260 nm of a DNA solution increases when thedouble helix is separated into single strands because of the bases unstack.5.Ribozyme （核酶）are the RNA molecules with catalytic activity. The activity of these ribozymes ofteninvolves the cleavage of a nucleic acid.6.De novo synthesis (从头合成)De novo synthesis of nucleotides begins with their metabolic precursors:amino acids, ribose-5-phosphate, one carbon units, CO2. mostly in liver.7.Salvage pathways （补救合成）Salvage pathways recycle the free bases and nucleosides released fromnucleic acid breakdown. Mostly in brain and marrow.8.Semi-conservative replication (半保留复制）DNA is synthesized by separation of the strands of aparental duplex, each then acting as a template for synthesis of a complementary strand based on the base-paring rule. Each daughter molecule has one parental strand and one newly synthesized strand. 9.Telomere（端粒）：Specialized structure at the end of a linear eukaryotic chromosome, which consists ofproteins and DNA, tandem repeats of a short G-rich sequence on the 3 ' ending strand and its complementary sequence on the 5' ending strand, allows replication of the extreme 5' ends of the DNAwithout loss of genetic information and maintains the stability of eukaryote chromosome.10.Telomerase（端粒酶）An RNA-containing reverse transcriptase that using the RNA as a template, addsnucleotides to the 3 ' ending strand and thus prevents progressive shortening of eukaryotic linear DNA molecules during replication.11.Reverse transcription (逆转录）Synthesis of a double-strand DNA from an RNA template.12.Reverse transcriptase (逆转录酶）A DNA polymerase that uses RNA as its template.activity: RNA-dependent DNA polymerase; RNAse H;DNA-dependent DNA polymerase13.The central dogma (中心法则）It described that the flow of genetic information is from DNA to RNA andthen to protein. According to the central dogma, DNA directs the synthesis of RNA, and RNA then directs the synthesis of proteins.14.asymmetric transcription（不对称转录）1..Transcription generally involves only short segments of aDNA molecule, and within those segments only one of the two DNA strands serves as a template.2.The template strand of different genes is not always on the same strand of DNA. That is, in anychromosome, different genes may use different strands as template.15.template strand （模板链）The DNA strand that serves as a template for transcription. (The relationshipbetween template and transcript is base paring and anti-parallel)16.non-template strand (or coding strand)（编码连）The DNA strand that opposites to the templatestrand.(Note that it has the same sequence as the synthesized RNA, except for the replacement of U with T )17.promoter i s the DNA sequence at which RNA polymerase binds to initiate transcription. It is alwayslocated on the upstream of a gene.18.Split genes (断裂基因)Split genes are those in which regions that are represented in mature mRNAs orstructural RNAs (exons) are separated by regions that are transcribed along with exons in the primary RNA products of genes, but are removed from within the primary RNA molecule during RNA processingsteps (introns).19.Exon(外显子) can be expressed in primary transcript and are the sequences that are represented inmature RNA molecules, it encompasses not only protein-coding genes but also the genes for various RNA (such as tRNAs or rRNAs)20.Intron（内含子）can be expressed and be the intervening nucleotide sequences that are removed fromthe primary transcript when it is processed into a mature RNA.21.Spliceosome（剪切体）A multicomponent complex contains proteins and snRNAs that are involved inmRNA splicing.22.Translation（翻译）The process of protein synthesis in which the genetic information present in anmRNA molecule (transcribed from DNA) determines the sequence of amino acids by the genetic codons.Translation occurs on ribosomes.23.genetic codon（密码子）The genetic code is a triplet code read continuously from a fixed starting pointin each mRNA, also called triplet. Genetic code defines the relationship between the base sequence of mRNA and the amino acid sequence of polypeptide.24.Degeneracy of code（密码子简并性）One codon encodes only one amino acid;More than 2 codons can encode the same amino acid;Most codons that encode the same amino acid have the difference in the third base of the codon.25.ORF（开放阅读框架）The nucleotideacids sequences in mRNA molecule from 5’AUG to 3’stop codon(UAA UAG UGA). It consists of a group of contiguous nonoverlapping genetic codons encoding a whole protein. Usually, it includes more than 500 genetic codons.26.Shine-Dalgarno sequence（SD）is a sequence upstream the start codon in prokaryotic mRNA that canbase pairs to a •UCCU•sequence at or very near the 3' end of 16S rRNA, thereby binding the mRNA and small ribosomal subunit by each other.27.Polyribosome（多聚核糖体）Ribosomes(10~100) are tandemly arranged on one mRNA and move in thedirection of 5’to 3’.Such a complex of one mRNA and a number ofribosomes is called polyribosome.28.signal peptide（信号肽）It is a short conservative amino terminal sequence (13~36AA) that exists ona newly synthesized secretory protein. It can direct this protein to a specific locationwithin the cell. It is subsequently cleaved away by signal peptidase; also called signal sequence and targeting sequence.29.Operon（操纵子）: Bacteria have a simple general mechanism for coordinating the regulation of geneswhose products are involved in related processes: the genes are clustered on the chromosome and transcribed together. Most prokaryotic mRNAs are polycistronic. The single promoter requi red to initiate transcription of the cluster is the point where expression of all of the genes is regulated. The gene cluster, the promoter, and additional sequences that function in regulation are together called an operon. Operons that include 2 to 6 genes transcribed as a unit are common; some operons contain 20 or more genes.30.Housekeeping gene（管家基因）Genes that are expressed at a fairly consistent level throughout the cellcycle and from tissue to tissue. Usually involved in routine cellular metabolism. Often used for comparison when studying expression of other genes of interest.31.Trans-acting factors（反式作用因子）：Usually considered to be proteins, that bind to the cis-actingsequences to control gene expression. The properties of different trans-acting factors:subunits of RNA polymerasebind to RNA Polymerase to stabilize the initiation complexbind to all promoters at specific sequences but not to RNA Polymerase (TFIID factor which binds to the TATA box)bind to a few promoters and are required for transcription initiation32.Cis-acting elements（顺式作用元件）：DNA sequences in the vicinity of the structural portion of a genethat are required for gene expression. The properties of different cis-acting elements:contain short consensus sequencesmodules are related but not identicalnot fixed in location but usually within 200 bp upstream of the transcription start sitea single element is usually sufficient to confer a regulatory responsecan be located in a promoter or an enhancerassumed that a specific protein binds to the element and the presence of that protein is developmentally regulated33.Southern blotting：Genomic DNA (from tissues or cells) are cut by RE, separated by gelelectrophoresis and denatured in solution, then transferred to a nitrocellulose membrane for detecting specific DNA sequence by hybridization to a labeled probe. It can be used to quantitative and qualitative analyze genomic DNA, or analyze the recombinant plasmid and bacteriophage (screening DNA library).34.Northern blotting: RNA samples (from tissues or cells) are separated by gel electrophoresis anddenatured in solution, then transferred to a nitrocellulose membrane for detecting specific sequence by hybridization to a labeled probe. It can be used to detect the level of specific mRNA in some tissues (cells) and to compare the level of same gene expression in different tissues (cells) or at different development period.35.Western blotting：rotein samples are separated by PAGE electrophoresis, then electro-transferred to NCmembrane. The proteins on NC membrane hybridize with a specific antibody (1st antibody ), then the target protein binding with antibody is detected with a labeled secondary antibody (2nd antibody).Also called immunoblotting. It can be used to detect the specific protein, semi-quantify specific protein, etc.36.PBlotting technique（印迹）:Transfer (blot) biological macromolecules separated in the gel and fix themto nitrocellulose/nylon membrane by diffusion, electro-transferring or vacuum absorption, then detectit.37.Nucleic acid probe（探针）:DNA or RNA fragment labeled with radioisotope, biotin orfluorescent, is used to detect specific nucleic acid sequences by hybridization38.PCR: PCR is a technique for amplifying a specific DNA segment in vitro. The reaction system includeDNA template, T aq DNA pol, dNTP，short oligonucleotide primers, buffer containing Mg2+. The process including 3 steps: denature, annealing, extension39.DNA coloning（克隆）:T o clone a piece of DNA, DNA is cut into fragments using restriction enzymes. Thefragments are pasted into vectors that have been cut by the same restriction enzyme to form recombinant DNA. The recombinant DNA are needed to transfer and maintain DNA in a host cell. This serial process and related technique are called DNA coloning or genetic engineering.40.Genomic DNA library(基因组DNA文库) A genomic library is a set of clones that together representsthe entire genome of a given organism. The number of clones that constitute a genomic library depends on (1) the size of the genome in question and (2) the insert size tolerated by the particular cloning vector system. For most practical purposes, the tissue source of the genomic DNA is unimportant because each cell of the body contains virtually identical DNA (with some exceptions).41.cDNA library（cDNA文库）:A cDNA library represents a sample of the mRNA purified from a particularsource (either a collection of cells, a particular tissue, or an entire organism), which has been converted back to a DNA template by the use of the enzyme reverse transcriptase. It thus represents the genes that were being actively transcribed in that particular source under the physiological, developmental, or environmental conditions that existed when the mRNA was purified.42.α-complementation（α互补）:Some plasmid vectors such as pUC19 carry the alpha fragment of the lacZ gene. The alpha fragment is the amino-terminus of the beta-galactosidase. Typically, the mutant E. coli host strain only carry the omega fragment, which is the carboxy-terminus of the protein. Either omegaor alpha fragment alone is nonfunctional. When the vector containing lac Z introduced into mutant E.coli, both the alpha and omega fragments are present there is an interaction and a functionally intact beta-galactosidase protein can be produced. This interaction is called alpha complementation.43.Secondary messenger(第二信使) are some small signal molecules that are generated in the cell inresponse to extracellular signals. They can activate many other downstream components. The most important second messengers are: Ca2+, cAMP, cGMP, DAG, IP3, Cer, AA and its derivatives, etc.44.Adaptor protein（衔接蛋白）A specialized protein that links protein components of the signalingpathway, These proteins tend to lack any intrinsic enzymatic activity themselves but instead mediate specific protein-protein interaction that drive the formation of protein complexes.45.Scaffolding protein（支架蛋白）A protein that assembles interacting signaling proteins intomultimolecular, it recruits downstream effectors in a pathway and enhances specificity of the signal. 46.Oncogene（癌基因）A gene whose product is involved either in transforming cells in culture or ininducing cancer in animals including virus oncogene（v-onc）and cellular-oncogene（c-onc ）。
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Why?
a
10
Outline
2020/8/ 18
Topic 1-4: Four components of translation machinery
mRNA
tRNA
attachment of amino acids to tRNA (aminoacyl-tRNA synthetases)
What is the key attribute of each component?
How the four component work together to accomplish translation?
reductionism(还原论) holism (整体论)
philosophy
a
13
stop codon
5’-AUGA-3’
start codon
ORF1
ORF2
ORF3
overlapping
a
28
Finding of Ribosome binding site (RBS) or SD-sequence
Scientific question: how can the cell recognize the correct start codon ?
AUG
AUG
AUG
start codon ordinary codon
Model: R17 phage infects E. coli
R17 phage: three genes—coding for A protein, coat protein and replicase (复制酶）
Phenomenon: 生物学现象
The revised central dogma 18
基
DNA repair and recombination
因
组
的
保 持
基
RNA processing
因 组
的
Translation
表 达
a
6
What is translation?
2020/8/ 18
--it is the story about decoding the genetic information contained in messenger RNA (mRNA) into proteins
a
16
2020/8/
1-1 polypeptide chains 18 are specified by ORF
open reading frame (ORF,开放阅读框) : a contiguous, nonoverlapping string of codons
Each ORF specifies a single protein and begins with a start codon and ends with a stop codon
2020/8/ 18
Functions: 1.Defines the end of ORF
2.Signal termination of polypeptide synthesis
a
20
2020/8/ 18
open reading frame (ORF): a contiguous stretch of codons “read” in a particular frame (decided by start codon)
2020/8/ 18
Welcome to My Molecular Biology
Class
Yang Yang（杨 洋 教授）
Huazhong University of Sciences and Technology
a
1
2020/8/
Molecular Biology of the Gen18 e,
genetic information: nucleotides in messenger RNA (mRNA)
protein: linear sequences of amino acids in protein
a
7
2020/8/ 18
The language of mRNA: four alphabet (A,G,C,U) translation
“open” to translation because it lacks a stop codon (until the last codon in ORF)
a
21
Eukaryotic mRNA (真核生物mRNA) monocistrionic, 单顺反子
2020/8/ 18
mRNA polypeptide
6/E --- Watson et al. (2008)
Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods
ribosome
Topic 5-7: Translation process
initiation; elongation; termination Topic 8: Antibiotics (抗生素）and
translation
a
11
2020/8/ 18
Basic machinery of Translation
30S subunit （ 核糖体30S 小亚基）is the key factor determining the translation
? ?
30S subunit was further dissociated into RNA and protein
How does the ribosome orchestrate the translation process?
a
14
2020/8/ 18
Topic 1: mRNA(信使RNA)
DNA
mRNA
transcription
3-nt-long codons (三联体密码子) specify the order of amino acids
mRNA：5-AUGAGUAACGCG-3 translation
Protein： Met-Ser-Asn-Ala
a
9
2020/8/ 18
Translation extremely costs
In rapid growing bacterial cells, protein synthesis consumes
2020/8/ 18
1-2 Prokaryotic mRNAs
have a ribosome binding site (核糖体结合位点)that
recruits the translational machinery(核糖体)
a
25
Ribosome
binding
site
(RBS)
or
SD-2020/8/ 18
a
15
2020/8/ 18
DNA： 5-ATGAGTAACGCG-3 Nontemplate strand 3- TACTCATTGCGC-5 template strand transcription
mRNA：5-AUGAGUAACGCG-3 translation
Protein： Met-Ser-Asn-Ala
Questions addressed in “Basic machinery of Translation”
2020/8/ 18
What is the organization of nucleotide sequence information in mRNA?
What is the structure of tRNAs? How do aminoacyl tRNA synthetases recognize and attach the correct amino acids to each tRNA?
a
2
2020/8/ 18
Part III: Expression of the Genome
This part concerned with one of the greatest challenges in understanding the gene－ how the gene is expressed
E.coli （大肠杆菌）ribosome can translate all three phage genes in vitro(体外）
Ribosome from another bacterium Bacillus
（芽孢杆菌）can only translate the A protein gene
incorporated into the growing polypeptide chain
2.Defines the reading frame （阅读框）for all subsequent codons
a
18
start codon
2020/8/ 18
a
19
The stop codon （终止密码子） UAG, UGA, or UAA (5’-3’)
a
17
The start codon （起始密码子）
2020/8/ 18
—the first codon of an ORF