參考成果2大一下課件geneexpression

1、Gene expression and regulationZhaoyuan Hou, Ph. D.joe_2014.4.28Life cycle of human being The significance of understanding of gene expression and regulation1996-2003 3 SHE groupAlcohol metabolismEthanolADHAcetaldehydeAcetateALDHADH1B.1: CaucasianADH1B.2: ChineseADH1B.3: IndianALDH1 (cytoplasm)ALDH2

2、(Mitochondria)50% of Chinese, Japanese do not have ALDH2 activitySix Steps at which eucaryotic gene expression are controlled 1.1 ConceptsGene: A DNA segment that contains the all genetic information required to encodes RNA and protein molecules. Genome: A complete set of genes of a given species. G

3、ene expression: A process of gene transcription and translation. Specificity of gene expression Temporal specificity (also called stage specificity): why sets of genes are expressed in the infants, not in the aged ones? Spatial specificity (also called tissue specificity): why in liver, not in brain

4、? Specificity of gene expression Paddock S.W. (2001). Paddock S.W. (2001). BioTechniquesBioTechniques 30: 756 - 761. 30: 756 - 761. Types of gene expressionConstitutive expressionSome genes are essential and necessary for life, and therefore are continuously expressed, such as those enzymes involved

5、 in TCA. These genes are called housekeeping genes. Induced or repressed genesThe expression levels of some genes fluctuate in response to the external signals. Some genes demonstrate higher expression level once being activated. It is called induced expression. On the other hand, some genes are rep

6、ressed and their expression levels are lower. It is called repressed expression. 1.2 Regulatory Elements Gene expression is a multiple-level process. Transcription initiation is a key point to control gene expression.Basic elements that regulate the transcription include: a. Special DNA sequencesb.

7、Regulatory proteinsc. DNA-protein interaction and protein-protein interactiond. RNA polymerase For prokaryotic systems: Operon is composed of structural genes, promoter, operator, and other regulatory sequences. a. Special DNA sequenceOther requlatory sequenceOperatorPromoter Sturctural genespromote

8、roperatorstructural generepressorRNA polThe DNA sequence adjacent to the structural genes that the repressor protein can bind to and prevent the transcription of structural genes. Operator structural genestartABCis-acting elements is the special DNA sequence that can affect the expression of its own

9、 gene. For eukaryotic systems:b. Regulatory proteinsFor prokaryotic systems: Specificity factor: It facilitates the binding of RNA-pol to particular DNA sequence, e.g. Sigma factor Repressor: It binds to the operator and prevent the transcription, known as negative regulation. Activator: It associat

10、es with DNA near the initiation point, resulting in the increase of RNA-pol binding affinity and the enhancement of the transcription efficiency. 5 major ways of gene repressor protein to be functional For eukaryotic systems: The regulatory proteins are called transcription factors (TF). After expre

11、ssion, TF will interact with the cis-acting elements to activate another genes. Therefore, they are referred to as trans-acting factors. Trans-acting factorsaADNAmRNAprotein AbA The regulation is implemented through numerous interactions between cis-acting elements and trans-acting factors. They are

12、 non-covalent bond. c. DNA-protein interactions Proteins may interact with each other prior to the DNA binding. Proteins can form a homo or hetero-dimer to function properly. Present in prokaryotes as well as eukaryotes.Protein-protein interactionsSection 2Gene Regulation in Prokaryote Common featur

13、es Prokaryotic genes are polycistron systems, that is, several relevant genes are organized together to form one transcription unit - operon. The majority of gene regulation is negative. Inducers are used to remove the repression. Operons: A cluster of functionally related genes can be under coordin

14、ated control by a single “on-off switch” The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control The operon can be switched off by a protein

15、repressor The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase The repressor is the product of a separate regulatory gene The operon can be switched off by a protein repressor The repressor prevents gene transcription by binding to the operator and blockin

16、g RNA polymerase The repressor is the product of a separate regulatory geneRepressible and Inducible Operons: Two Types of Negative Gene Regulation A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription The trp operon is a repressible operon An

17、 inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription Lac operon is an inducible operon The lac operon contains genes that code for enzymes used in the hydrolysis and metabolism of lactose By itself, the lac repressor is activ

18、e and switches the lac operon off A molecule called an inducer inactivates the repressor to turn the lac operon onAYZOPIstructural genepermease-galactosidaseregulatory siteoperatorpromoterCAP-binding siteregulatory genetransacetylaseStructure of lac operonJacobMonod modelWinners of 1965 NP awardMeta

19、bolism of lactose Bacteria do not express these three enzymes when glucose is available. However, bacteria produce those enzymes if lactose is present and glucose is absent.Inducible expression Sequence of lac operon lac operon (TTTACA/TATGTT) is a basic promoter, and has a weak expression activity.

20、 CAP (Catabolite gene activator protein) binding site is at -60 region. CAP is a homodimer with binding ability to DNA and cAMP. Glucose inhibits the formation of cAMP. When glucose is present, cAMP is low. Only after glucose is exhausted, level of cAMP becomes higher. The CAP-cAMP complex is formed

21、, and this complex binds to the CAP binding site on lac operon to enhance pol II complex activity. AYZOPImRNARNA polAt normal condition, E. coli utilizes glucose as energy source. Lactose is absent, no lac gene is expressed. Situation 1 lacI gene has its own promoter, and its expression can produce

22、LacI repressor. The tetrameric Lac repressor binds to the lac operator site Olac. The binding blocks the RNA-pol moving on DNA template, and no lacZ, lacY, and lacA are expressed. AYZOPIrepressormRNAlactoseallolactoseRNA polgalactosidaseSituation 2When glucose and lactose are both present, lacZ, lac

23、Y, and lacA genes are weakly expressed. The E. coli still prefers to use glucose. The galactosidase is weakly expressed (at the basal level). When lactose is present, it is converted to allolactose or galactose that binds to the repressor. The repressor can no longer bind to the operator, and lac ge

24、ne can be expressed. Galactose , Allolastose and IPTG are referred to as inducers. Inducers The lacZYA RNA transcript is very unstable and could be degraded quickly. Therefore, the synthesis of three enzymes will be cease under normal condition. Presence of lactose AYZOPICAPcAMPRNA polWhen glucose i

25、s absent and lactose is present, the CAP-cAMP complex binds to the CAP site to activate the lac gene. Situation 3 OOOORNA pollactoseno lactoseRNA polRNA polRNA pol Coordinate expressionNo glucoseGlucose By default the trp operon is on and the genes for tryptophan synthesis are transcribed When trypt

26、ophan is present, it binds to the trp repressor protein, which turns the operon off The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are highTrp operonPromoterDNARegulatory genemRNAtrpR5 3 ProteinInactive r

27、epressorRNApolymerasePromotertrp operonGenes of operonOperatormRNA 5 Start codonStop codontrpEtrpDtrpCtrpBtrpAEDCBAPolypeptide subunits that make upenzymes for tryptophan synthesis(a)Tryptophan absent, repressor inactive, operon on(b) Tryptophan present, repressor active, operon offDNAmRNAProteinTry

28、ptophan (corepressor)Activerepressor(b) Tryptophan present, repressor active, operon offDNAmRNAProteinTryptophan (corepressor)ActiverepressorNo RNAmade The trp repressor gene can be expressed, but it does not bind to the operator. When Trp is more than enough, the repressor will form a complex with

29、Trp. The complex binds to the operator, blocking the synthesis of Trp. Application:Large scale production of protein from E. coli1. IPTG is a molecular mimic of allolactose, a lactose metabolite. 2. Can bind to the lac repressor and induce the lac operon transcription3. Can not be degradated by beta

30、-galactosidase 4. IPTG uptake by E. coli is independent of lactose permease Section 3Regulation of Eukaryotic TranscriptionChromatin remodeling Structural featuresLarge genome: 3 x 109 bps, 35 000 genesMonocistronRepeated sequences: different lengths and different frequencies. Often inverted repeats

31、Splite genes: separated by introns and exons alternatively Regulation features1. RNA-pol: 3 forms (I, II, and III) for different RNAs2. Changes of chromosomal structure Hypersensitive site Base modification Isomer-conversion Histone changes 3. Positive regulation4. Transcription and translation are

32、separated5. Post-transcriptional modification6. Regulation through intercellular and intracellular signals 3.1 Cis-acting elements They are specific DNA sequences, each of which regulates transcription of one or more genes. They usually have consensus sequences. Promoter: TATA box, CAAT box, and GC

33、box, Sequence: TATAAAA Location: - 25 - 30 bp Function: It is the binding site for TFII D, which is required for RNA polymerase binding. It controls the veracity and frequency of transcriptional initiation.TATA box Sequence: GCCAAT Location: -70 bp Function: It is the binding site for CTF1 (CAAT-bin

34、ding transcription factor) and C/EBP (enhancer binding protein). CAAT box Sequence: GGGCGGLocation: -30 -110 bpFunction: It is the binding site for a protein called Sp1. GC box It is a DNA sequence that can determine the temporal and spatial specificities of expression and increase the promoter acti

35、vity. enhancer It is a negative regulation element. It will repress the transcription once interacted with specific proteins. Silencer 3.2. Trans-acting factors They are the proteins that bind indirectly to cis-acting elements and then regulate the transcription initiation. The trans-acting factors

36、can be transcription factors (TF). transcription factors General transcription factors Special transcription factors Transcription activators EBP (enhancer binding protein) Transcription inhibitors General structure of TF DNA-binding domain Activation domain Protein-protein interaction domain Genera

37、l structure of TF CTD of RNA-pol II is an important point of interaction with mediators and other protein complexes. Cofactors facilitate the TF assembly. 3.3 DNA-protein interactions Regulatory proteins have discrete DNA-binding domains of particular structure, i.e., binding motif. The AA side chai

38、ns of regulatory proteins interact with bases of DNA through H bonds. Yeast activator protein GCN4Leucine zipperZinc fingerSteroid hormone receptor Helix-loop-helix Helix-loop-helixHuman transcription factor MAX Helix-turn-helixLac repressor Helix-turn-helixTrp repressorSummaryLactose operonTypes of

39、 DNA binding motifs in eukaryotesReview of the contents from the 4 classes DNA RNA protein transcription translation replication reverse transcription Central dogmaDNA ReplicationExperiment of DNA semiconservative replicationHeavy DNA(15N)grow in 14N mediumThe first generationgrow in 14N mediumThe s

40、econd generationMany DNA fragments are synthesized sequentially on the DNA template strand having the 5 - end. These DNA fragments are called Okazaki fragments. They are 1000 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes. The daughter strand consisting of Okazaki fragments is called the lagging strand. Okazaki fragments The synthesis of RNA molecules using DNA str