基因表达调控

1、要点,知识点,1,真核细胞基因表达的调控主要发生,在四个相对独立的水平上,知识点,2,基因表达在转录水平上调控,知识点,3,加工水平的基因表达调控,知识点,4,翻译水平调控,知识点,5,翻译后水平调控,知识点,1,真核细胞基因表达的调控主要发生在四,个相对独立的水平上,名词,基因表达调控：细胞使遗传信息在正确时间正,确位置进行正确表达或关闭以行使特定功能的调,节机制。真核细胞基因表达调控发生在,4,个彼此相,对独立的水平上,1,转录水平：决定某个基因不否会被转录，什么时,候转录，转录的频率,2,加式水平：决定初始的,RNA,转录产物如何剪接和,加工为成熟的,mRNA,3,翻译水平：决定某种,m

2、RNA,是否会真正得到翻译,如果能得到翻译，决定翻译的频率和时间,4,翻译后水平：在蛋白被,翻译后，选择性激活蛋,白或使蛋白失活,知识点,2,基因表达在转录水平上调控,通用转录因子与结合,RNA,聚合酶的核心启动子位点,结合，起始基础水平的转录。特异转录因子分为,激活基因转录的激活型转录因子和抑制转录的抑,制型转录因子，与基因的各种调控位点结合，促,进或抑制基因转录。激素通过激活或抑制某些特,异转录因子（激素受体）而影响基因转录。转录,因子通常通过调节组蛋白核心结构而改变核小体,和染色质紧密程度，影响通用转录因子和,RNA,聚,合酶对启动子的结合来调节基因表达。激活型转,录因子通常有利于导致染

3、色质或组蛋白结构松散,的蛋白质发挥作用，如组蛋白乙酰化酶；抑制型,转录因子通过会加强促进染色质结构紧密的蛋白,质作用，如组蛋白脱乙酰酶,Eukaryotic transcription of,protein-encoding genes is a highly regulated process in which,numerous transcription activators recognize specific DNA elements, proximal and distant to,promoters, and direct assembly of,general transcrip

4、tion factors, co-activator complexes and,RNA polymerase II on promoters (the pre-initiation complex, at least 50 polypeptides). To,understand the molecular mechanism of,activator-dependent transcription initiation, one needs,to understand the pathway of,assembly of,the pre-initiation complex, the st

5、ructural,organization of,the pre-initiation complex, and how the assembly and structure of,the pre,initiation complex are affected by binding of,activators to cognate DNA elements,a very,challenging task for traditional, ensemble, in vitro, methods,Model of hormone-dependent gene activation by the g

6、lucocorticoid,receptor (GR,In the absence of,hormone,GR is bound in a complex with Hsp90 in the,cytoplasm,via its,ligand,binding,domain,light purple). When,hormone,is present, it diffuses through the,plasma membrane,and binds to the GR,ligand,binding,domain,causing a conformational change in the,lig

7、and,binding,domain,that releases the,receptor,from Hsp90. The,receptor,with bound,ligand,is then translocated into the,nucleus,where its DNA,binding,domain,orange) binds to response elements, allowing the,activation,domain,green) to stimulate,transcription,of target,genes,The nuclear receptor superf

8、amily,All nuclear hormone receptors bind to DNA as either homodimers or heterodimers, but,for simplicity we show them as monomers here. (A) The receptors all have a related,structure. The short DNA-binding domain in each receptor is shown in,green,B) A,receptor protein in its inactive state is bound

9、 to inhibitory proteins. Domain-swap,experiments suggest that many of the ligand-binding, transcription-activating, and DNA,binding domains in these receptors can function as interchangeable modules. (C) The,binding of ligand to the receptor causes the ligand-binding domain of the receptor to,clamp

10、shut around the ligand, the inhibitory proteins to dissociate, and coactivator,proteins to bind to the receptors transcription-activating domain, thereby increasing,gene transcription. (D) The three-dimensional structure of a ligand-binding domain with,right,and without,left,ligand bound. Note that

11、the,blue,helix acts as a lid that snaps,shut when the ligand (shown in,red,binds, trapping the ligand in place,知识点,3,加工水平的基因表达调控,名词,mRNA,剪接：由于真核细胞基因为不连续基因，包含编,码的外显子和不编码的内含子，转录时都被转录，之后将,不编码的内含子和不需要的外显子切除，形成成熟的,mRNA,这个过程称为,mRNA,剪接,剪接有,2,种基因方式,1,组成型剪接,2,选择性剪接,名词,组成型剪接：将前体,mRNA,中的内含子剪除，规范地将,外显子拼接成成熟,mRN

12、A,一个基因通过组成型剪接只产,生一种成熟,mRNA,名词,选择性剪接：不仅将内含子剪除，还将外显子选择性,剪除，从外显子按照不同的方式拼接在一起。一个基因通,常可通过选择性剪接产生多个成熟,mRNA,翻译产物氨基,酸序列有差别，差别程度有大有小。剪接增强子位于保留,与否受到调节的外显子内，当它与调节蛋白结合，这个外,显子保留；当它不与调节蛋白结合，则被剪除,Types of,alternative splicing,In these,graphics, exons are represented by boxes,and introns by lines. Exon regions,incl

13、uded in the messages by alternative,splicing are colored while constitutive,exons are shown in gray. Promoters are,indicated with arrows and,polyadenylation sites with AAAA,Figure 2. Locations of,regions on the pre-mRNA that can affect alternative,splicing,Some combination of,these regulatory region

14、s can usually be found,Weaker consensus splice sites surrounding the alternative exon, exonic regulatory,regions and intronic regulatory regions are indicated,From research over the past 20 years, some general themes have emerged for,alternative splicing regulation, although the exact mechanisms sti

15、ll need to be,determined. Alternatively spliced exons often have weak consensus sequences at,the 5 and 3 ends of,the introns, suggesting that additional signals are required for,recognition of,the exon by the splicing machinery. -acting pre-mRNA sequences,responsible for regulation of,splicing have

16、been identified for many genes. These,regions are found in exons or in introns and can be enhancers or silencers of,splice,site usage,Figure 2,. These sequence motifs serve as binding sites for protein,factors that can enhance or inhibit the ability of,the spliceosome to recognize the,exons. The exo

17、nic elements not only encode amino acids but also regulate their,own ability to be spliced into the mature message. -acting splicing factors that,interact with splicing regulatory elements in exons have been identified. Subsets of,the SR proteins bind with regulatory sequences important for splicing

18、 control,Heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family members can bind,to high-affinity sequences in exons and inhibit splicing through blocking SR,proteins from binding to the exons,Alpha,原肌球蛋白基因转录产物在不同的细,胞中有不同的剪接形式,在横纹肌细胞,在平滑肌细胞,在成纤维细胞,在纤维细胞,在肝细胞,在脑细胞,原肌球蛋白,初始,MRA,选择性拼接产生不同的成熟,mRNA,D

19、scam is a member of the immunoglobulin superfamily and contains,10 Ig-domains, 6 fibronectin type III (FNIII) domains, a single,transmembrane domain, and a novel 374 AA containing cytoplasmic,domain. The Dscam mRNA comprises 24 exons. Tandem arrays of,alternative exons 4, 6, 9 and 17 allow the gener

20、ation of variable Ig,domain sequences for Ig 2, 3 &7 and two different transmembrane,domains. Mutually exclusive splicing occurs for exons 4, 6, 9, and 17,Genomic and cDNA analysis revealed the potential of generating,some 38,000 isoforms of Dscam. Variable exons are shown in color,Gray lines in gen

21、omic DNA and boxes in mRNA represent constant,exons,Dietmar Schmucker, Ph.D,Associate Professor of Neurobiology,Harvard Medical School,Department of Cancer Biology,Dana-Farber Cancer Institute,My laboratory uses the model organism Drosophila,fruit fly) to study molecular mechanisms that control,the

22、development of neuronal connectivity. Any,assembly of neuronal circuits, simple or complex, is,controlled by a series of specific molecular signaling,systems that instruct the directional growth of neuronal,processes (axons and dendrites). We combine genetic,biochemical and cell biological approache

23、s to study,how neuronal surface receptors control these signaling,systems during axon and dendrite guidance, branching,and synaptic target selection. We focus our,developmental and functional studies primarily on the,analysis of sensory neurons of the visual and somato,sensory system of flies,RNA,的可

24、变剪接有时会产生功能完全没有联系的蛋白质,甲状腺中经选择性剪,接，翻译产物是降钙,素,calcitonin,下丘脑中，经选择性剪接，产物是,降钙素基因相关多肽,GGRP,与,血压的调节和痛觉有关,选择性拼接产生不同的成熟,mRNA,知识点,4,翻译水平调控,mRNA,与多种蛋白质作用，决定是否被翻译和翻译的速度,翻译的时间,1,如果,mRNA,与抑制蛋白结合，就会失去活性而不被翻译,如未受精卵细胞中的,mRNA,与抑制性蛋白结合形成“隐,蔽,mRNA,或者通过,microRNA,和,siRNA,途径降解,mRNA,使得,mRNA,得不到翻译,2,非特异性翻译速度调节机制：影响所有,mRNA,的

25、,翻译，如,翻译起始因子,eIF2,磷酸化，降低起始翻译的活性，从而减,慢蛋白质合成速率,3,特异性翻译速率调节机制：只改变特定,mRNA,的翻译速率,如编码铁蛋白,mRAN,的翻译速率的调控,4,翻译时间的调控：通过调节,mRNA,的稳定性而调节翻译的,时间，如转铁蛋白受体,mRNA,半寿期的调节,5,翻译的空间调控：有些,mRNA,在,3,端非翻译区有细胞质定,位信息，通过微管和微丝运输并定位在细胞特定区域,Model of CPEB-dependent masking and,activation of mRNA translation in oocytes,Typically, mat

26、ernal mRNAs initially carry a,short poly(A) tail and their CPE motif is,bound by CPEB. CPEB forms a complex with,maskin, which in turn interacts with eIF4E,This configuration is thought to represent a,translationally inactive or “masked” state of,the mRNA. The signal for oocyte maturation,leads to a

27、 phosphorylation of CPEB and a,stimulation of binding between CPEB and,CPSF. Through further association of CPSF,with poly(A)-polymerase, this leads to,elongation of the poly(A) tail (indicated by,the line with star). The binding between,maskin and eIF4E is reduced, possibly as a,consequence of poly

28、adenylation. This clears,the way for efficient recruitment of eIF4G,through binding to eIF4E and PABP and,activation of translation (indicated by a,further line with star,By,RNA interference,short RNAs can lead,to silencing the expression of genes that,contain complementary sequences in their,mRNA,A

29、 compex of double-stranded RNA is,cleaved into short fragments of 21-22,basepairs in length by the ribonuclease,Dicer,The fragments are siRNAs (short,interfering RNAs,The siRNAs bind to the RISC (RNA,induced silencing complex,One of the strands of siRNA is degraded,The remaining single-stranded siRN

30、A,complexed with the RISC can then bind to,complementary mRNA,If a perfect or near perfect match, the,mRNA is cleaved,In additon, the RISC-siRNA complex can,enter the nucleus, binds the genomic,sequence and initiates a DNA methylation,based chromatin condensation inactivation,of the gene,In a relate

31、d mechanism,microRNAs,miRNAs) are gene products (mRNAs,that are 21-22 nucleotides in length,The primary miRNAs are transcribed,form hair-pin structures and are cleaved by,Drosha to make precursor microRNAs,roughly 70 nucleotides in length,The pre-miRNAs are exported to the,cytoplam where they are cl

32、eaved by dicer,into the 21-22 nucleotide mature,microRNAs,The miRNAs form ribonucleoprotein,complexes with mRNAs,If,the match is exact, the mRNA is,destroyed, similar to siRNA mechanisms,If,the match is less-than-exact, then,binding (usually of,several miRNAs,inhibit translation,Genes for miRNAs see

33、m to make up 0.5,1.0% of,the total number of,genes in,multicellular organisms,i.e. 200-250 miRNA genes in humans,The translation initiation factor eIF2 is a heterotrimeric complex that is responsible for,binding the initiator methionyl tRNA (Met-tRNAiMet) to the small ribosomal subunit. The g,subuni

34、t of,eIF2 contains a classic GTP-binding domain, and GTP-binding is essential for,binding Met-tRNAiMet to form the ternary complex of,eIF2, GTP and Met-tRNAiMet,During the course of,translation initiation the GTP bound by eIF2 is hydrolyzed to GDP,and eIF2 is released from the ribosome in a binary c

35、omplex with GDP,As eIF2 has a much,higher affinity for binding GDP than GTP,a guanine-nucleotide exchange factor (GEF,termed eIF2B is required to recycle eIF2,GDP to eIF2,GTP,In response to environmental stresses, a family of protein kinases phosphorylate eIF2 (eukaryotic initiation,factor 2) to all

36、eviate cellular injury or alternatively induce apoptosis. Phosphorylation of eIF2 reduces global,translation, allowing cells to conserve resources and to initiate a reconfiguration of gene expression to,effectively manage stress conditions. Accompanying this general protein synthesis control, eIF2,p

37、hosphorylation induces translation of specific mRNAs, such as that encoding the bZIP (basic leucine zipper,transcriptional regulator ATF4 (activating transcription factor 4). ATF4 also enhances the expression of,additional transcription factors, ATF3 and CHOP (CCAAT/enhancer-binding protein homologo

38、us,protein)/GADD153 (growth arrest and DNA-damage-inducible protein), that assist in the regulation of genes,involved in metabolism, the redox status of the cells and apoptosis. Reduced translation by eIF2,phosphorylation can also lead to activation of stress-related transcription factors, such as N

39、F-kB (nuclear,factor kB), by lowering the steady-state levels of short-lived regulatory proteins such as IkB (inhibitor of NF,kB). While many of the genes induced by eIF2 phosphorylation are shared between different environmental,stresses, eIF2 kinases function in conjunction with other stress-respo

40、nse pathways, such as those regulated by,mitogen-activated protein kinases, to elicit gene expression programmes that are tailored for the specific,stress condition. Loss of eIF2 kinase pathways can have important health consequences. Mice devoid of the,eIF2 kinase GCN2 general control non-derepress

41、ible-2 or EIF2AK4 (eIF2a kinase 4) show sensitivity to,nutritional deficiencies and aberrant eating behaviours, and deletion of PEK pancreatic eIF2a kinase or,PERK (RNA-dependent protein kinase-like endoplasmic reticulum kinase) or EIF2AK3 leads to neonatal,insulin-dependent diabetes, epiphyseal dys

42、plasia and hepatic and renal complications,The intracellular localisation of mRNAs,is a general mechanism to target proteins,to the regions of a cell where they are,required, and plays an important role in,the polarisation of many cell types. A,striking example of this phenomenon is,provided by the,

43、Drosophila,oocyte,where the localisation of,bicoid,oskar,and,gurken,mRNAs to three distinct,positions within the cell determines the,polarity of the anterior-posterior and,dorsal-ventral axes of the embryo. Using,the powerful genetics of,Drosophila,we,are using a combination of molecular,cell-biolog

44、ical and genetic techniques to,investigate the mechanism of mRNA,localisation. In addition, we are studying,how the two axes of the oocyte become,polarised to define the destination of,these transcripts, in order to understand,the origin of polarity in,Drosophila,development,The drosophila is a type

45、 of,fruit fly, a well-established,genetic model,Translation of,ferritin is activated in the presence of,iron,Translation is inhibited by binding of,the IRE-binding protein to the hairpin structure of,an,iron response element (IRE,in the 5 prime untranslated leader sequence of,ferritin,mRNA,When iron

46、 binds to IRE-binding protein, it contorts into a conformation that does not,recognize the IRE,When iron is available, ribosomes can assemble on the mRNA and proceed to translate,ferritin,The hairpin does not interfere with the ribosome activities,Degradation of,the,transferrin receptor mRNA,required for iron uptake) is also,regulated by the allosteric IRE-binding protein,Transferrin receptor mRNA has an IRE in its 3 prime untranslated region,When intracellular iron is low, the IRE-binding protein remains