三国大学脑 基因表达调控研究方法 Techniques of gene expression and requlation 生物化学与分子生物学教研室 连继勤 2015.9 Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 1 Techniques of gene expression and regulation 基因表达调控研究方法 生物化学与分子生物学教研室 连继勤 2015.9
Genes can be regulated at many levels DNA RNA PROTEIN TRANSCRIPTION TRANSLATION he"“ Central Dogma Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 2 Genes can be regulated at many levels DNA RNA PROTEIN TRANSCRIPTION TRANSLATION The “Central Dogma
Control of Gene Expression-Transcription Factors Transcription factors(TFS) are proteins that bind to the dna and help to control gene expression. We call the sequences to which they bind transcription factor binding sites(TFBSs), which are a type of CIS-regulatory sequence Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 3 Control of Gene Expression—Transcription Factors Transcription factors (TFs) are proteins that bind to the DNA and help to control gene expression. We call the sequences to which they bind transcription factor binding sites (TFBSs), which are a type of cis-regulatory sequence
Control of Gene Expression Transcription factor binding sites are found within larger functional units of the dNa called cis-regulatory elements There are two main type of cis-regulatory elements: promoters and cis-regulatory modules(sometimes calledenhancers) cis-regulatory module( CRM) TFBS Transcription factor binding site(TFBS) TFBS activators DNA O coactivator neral transcription ors RNA polymerase TATA box coding region RNA transcript Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 4 Control of Gene Expression Transcription factor binding sites are found within larger functional units of the DNA called cis-regulatory elements. There are two main type of cis-regulatory elements: promoters, and cis-regulatory modules (sometimes called “enhancers”). TFBS TFBS transcription factor binding site (TFBS) cis-regulatory module (CRM)
转录因子调控的基本方式 、通过与顺式调控元件相互作用影响转录活性 通过与其它转录因子或辅助蛋白相互作用影响转录活性 cis-regulatory module( CRM) TFBS Transcription factor binding site(TFBS) TFBS activators DNA O coactivator neral transcription ors RNA polymerase TATA box coding region RNA transcript Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 5 转录因子调控的基本方式 一、通过与顺式调控元件相互作用影响转录活性 二、通过与其它转录因子或辅助蛋白相互作用影响转录活性 TFBS TFBS transcription factor binding site (TFBS) cis-regulatory module (CRM)
研究基因表达调控的基本方法 、 DNARNA与蛋白质的相互作用技术 Report gene Assay EMSA a ChIP SElEX Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 6 研究基因表达调控的基本方法 一、DNA/RNA与蛋白质的相互作用技术 ❑ Report gene Assay ❑ EMSA ❑ ChIP ❑ SELEX
1. Reporter Gene Assay Measures the in vitro level of gene expression or transcriptional activity Transcription is initiated by binding of specific proteins to specific dna promoter regions Reporter assays use a specific DNA promoter fused to a reporter gene(luciferase, SEAP, GFP, etc.) Transcriptional activity of your promoter is measured y reporter expression Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 7 Measures the in vitro level of gene expression or transcriptional activity Transcription is initiated by binding of specific proteins to specific DNA promoter regions Reporter assays use a specific DNA promoter fused to a reporter gene (luciferase, SEAP, GFP, etc.) Transcriptional activity of your promoter is measured by reporter expression 1. Reporter Gene Assay
Control of Gene Expression: Promoters Every gene has a promoter the dna sequence immediately surrounding the transcription start site. The promoter is the site where RNa polymerase and the so-called general transcription factors bind coactivators general transcription factors RNA polymerase TAtA box coding region promoter RNA transcript Department of Biochemistry Molecular Biology e
Department of Biochemistry & Molecular Biology 8 Control of Gene Expression: Promoters Every gene has a promoter, the DNA sequence immediately surrounding the transcription start site. The promoter is the site where RNA polymerase and the so-called general transcription factors bind
Report gene Assay RNA polymerase and Transcription factors Reporter gene Promoter Transcription mRNA Translation Reporter Protein Department of Biochemistry Molecular Biology e 9
Department of Biochemistry & Molecular Biology 9 Report gene Assay
R eport gene 报告基因是一种编码可被检测的蛋白质或酶的 基因,是一个其表达产物非常容易被检测或鉴 定的基因。通常将包含基因启动子的DNA片断 置于一个无关的缺乏内源性启动子活性的报告 基因编码序列的上游,通过检测报告基因的活 性或表达水平来间接度量启动子的活性。 Department of Biochemistry Molecular Biology e 10
Department of Biochemistry & Molecular Biology 10 Report gene 报告基因是一种编码可被检测的蛋白质或酶的 基因，是一个其表达产物非常容易被检测或鉴 定的基因。通常将包含基因启动子的DNA片断 置于一个无关的缺乏内源性启动子活性的报告 基因编码序列的上游，通过检测报告基因的活 性或表达水平来间接度量启动子的活性