第五章 克隆基因的表达分析

有效转录的基本条件
启动子（promoter）：在基因序列中，标志着转录起始的可 被RNA聚合酶识别的位点(DNA区段)，一般位于基因的上游。 终止子（terminator）：位于基因的编码序列之外（一般在 下游）的一段标志着转录停止的RNA聚合酶识别位点。
Prokaryotic gene regulation differs from eukaryotic regulation, but since prokaryotes are much easier to work with, we focus on prokaryotes at this point. Promoters are sequences of DNA that are the start signals for the transcription of mRNA. Terminators are the stop signals. mRNA molecules are long (50010,000 nucleotides).
Translation is the process of converting the mRNA codon
sequences into an amino acid sequence. The initiator codon (AUG) codes for the amino acid N-formylmethionine (f-Met). No translation occurs without the AUG codon. f-Met is always the first amino acid in a polypeptide chain, although frequently it is removed after translation. The intitator tRNA/mRNA/small ribosomal unit is called the initiation complex. The larger subunit attaches to the initiation complex. After the initiation phase the message gets longer during the elongation phase.
第一节 基因表达的基本条件
1、基因表达的基本过程
2、有效转录的基本条件
3、正确翻译的基本条件
4、影响克隆基因表达效率的因素
转录的具体过程
Transcription(转录): making an RNA copy of a DNA sequence . RNA polymerase opens the part of the DNA to be transcribed. Only one strand of DNA (the template strand, antisense strand) is transcribed.
New tRNAs bring their amino acids to the open binding site on the ribosome/mRNA complex, forming a peptide bond between the amino acids. The complex then shifts along the mRNA to the next triplet, opening the A site. The new tRNA enters at the A site. When the codon in the A site is a termination codon, a releasing factor binds to the site, stopping translation and releasing the ribosomal complex and mRNA. Often many ribosomes will read the same message, a structure known as a polysome forms. In this way a cell may rapidly make many proteins.
《基因工程概论》
第一章 导 论
第二章 基因操作的工具酶
第三章 基因克隆的载体
第四章 基因克隆的策略
第五章 克Leabharlann Baidu基因的表达
第六章 基因工程的基本技术
第五章
克隆基因的表达
第一节 基因表达的基本条件
第二节 原核表达载体的构建 第三节 克隆基因在植物细胞中的表达
第一节 基因表达的基本条件
1、基因表达的基本过程
differs in each. The 30S unit has 16S rRNA and 21 different proteins. The 50S subunit consists of 5S and 23S rRNA and 34 different proteins. The smaller subunit has a binding site for the mRNA. The larger subunit has two binding sites for tRNA
正确翻译的基本条件
1、具备起始密码子 2、具备终止密码子
3、具有正确的阅读框
基 因 表 达 的 基 本 过 程
第一节 基因表达的基本条件
1、基因表达的基本过程
2、有效转录的基本条件
3、正确翻译的基本条件
4、影响克隆基因表达效率的因素
4、影响克隆基因表达效率的因素
一般而言，所用启动子的强度、DNA的转录其始序列、 密码子的选择、mRNA的二级结构、转录的终止、基因的拷贝 数等都会在一定程度上影响到转基因的表达。
第一节 基因表达的基本条件
1、基因表达的基本过程
2、有效转录的基本条件
3、正确翻译的基本条件
4、影响克隆基因表达效率的因素
正确翻译的基本条件
The Genetic Code
To code for the 20 essential amino acids a genetic code must consist of at least a 3-base set (triplet) of the 4 bases. If one considers the possibilities of arranging four things 3 at a time (4X4X4), we get 64 possible code words, or codons (a 3-base sequence on the mRNA that codes for either a specific amino acid or a control word). The genetic code was broken by Marshall Nirenberg and Heinrich Matthaei, a decade after Watson and Crick's work. The genetic code consists of 61 amino-acid coding codons and three termination codons, which stop the process of translation. The genetic code is thus redundant (degenerate in the sense of having multiple states amounting to the same thing), with, for example, glycine coded for by GGU, GGC, GGA, and GGG codons.
a. 启动子的结构对表达效率的影响
The genetic code
Messenger RNA (mRNA) is the blueprint for construction of a protein. Transfer RNA (tRNA) is the truck delivering the
proper amino acid to the site at the right time. tRNA will form a "cloverleaf" structure due to complementary base pairing. At the top of the large loop are three bases, the anticodon, which is the complement of the codon. There are 61 different tRNAs, each having a different binding site for the amino acid and a different anticodon.
转录的具体过程
Antisense strand
有效转录的基本条件
Groups of genes coding for related proteins are arranged in units known as operons. An operon consists of an operator, promoter, regulator, and structural genes. The regulator gene codes for a repressor protein that binds to the operator, obstructing the promoter of the structural genes. The regulator does not have to be adjacent to other genes in the operon. If the repressor protein is removed, transcription may occur.
半乳糖苷酶
渗透酶
转乙酰基酶
Operons are either inducible or repressible according to the control mechanism. Seventyfive different operons controlling 250 structural genes have been identified for E. coli. Both repression and induction are examples of negative control since the repressor proteins turn off transcription.
Ribosomes are the organelle (in all cells) where proteins are synthesized. They consist of two-thirds rRNA and one-third protein. Ribosomes consist of a small (in E. coli , 30S) and larger (50S) subunits. The length of rRNA
2、有效转录的基本条件
3、正确翻译的基本条件
4、影响克隆基因表达效率的因素
1、基因表达的基本过程
1、基因表达的基本过程
基因的表达主要涉及到两个过程：转录和翻译。 首先，目的基因通过转录(transcription)形成mRNA（信使 RNA, messenger RNA )； 然后， tRNA （转运 RNA, transfer RNA）将 各种氨基酸运送到核糖 体 并 按 照 mRNA 的 密 码 子顺序将各种氨基酸连 接成特定的氨基酸序列 (translation) 最终得到基 因的表达产物（蛋白 质）。