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Spatial specificity (also called tissue specificity): why in liver not in brain?PowerPoint Presentation: Specificity of gene expression: Type of gene expression a. Constitutive expression Some genes are essential and necessary for life, and therefore are continuously expressed, such as those enzymes involved in TAC. These genes are called housekeeping genes.: b. Induction and repression The expression levels of some genes fluctuate in response to the external signals.PowerPoint Presentation: Some genes demonstrate higher expression level once being activated. It is called induced expression. On the other hand, some genes are repressed 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 of controlling gene expression.Basic elements that regulate the transcription include: : Basic elements that regulate the transcription include: Special DNA sequences Regulatory proteins DNA-protein interaction and protein-protein interaction RNA polymerasePowerPoint Presentation: For prokaryotic systems: Operon is composed of structural genes , promoter, operator, and other regulatory sequences . a. Special DNA sequence Other requlatory sequence Operator Promoter Sturctural genes: The DNA sequence that RNA-pol can bind to and initiate the transcription. PromoterPowerPoint Presentation: The DNA sequence adjacent to the structural genes that the repressor protein can bind to and prevent the transcription of structural genes. Operator: Cis-acting elements is the special DNA sequence that can affect the expression of its own gene. For eukaryotic systems:PowerPoint Presentation: b. Regulatory proteins For prokaryotic systems: Specific factor: It facilitates the binding of RNA-pol to particular DNA sequence. Repressor: It binds to the operator and prevent the transcription, known as negative regulation.PowerPoint Presentation: Activator: It associates with DNA near the initiation point, resulting in the increase of RNA-pol binding affinity and the enhancement of the transcription efficiency.PowerPoint Presentation: For eukaryotic systems: The regulatory proteins are called transcription factors (TF). After expression, TF will interact with the cis-acting elements to activate another genes. Therefore, they are referred to as trans-acting factors .PowerPoint Presentation: Trans-acting factorsPowerPoint Presentation: The regulation is implemented through numerous interactions between cis-acting elements and trans-acting factors. They are non-covalent bond. c. DNA-protein interactionsPowerPoint Presentation: Proteins may have to interact with each other prior to the DNA binding. Proteins can form a homo or hetero-dimer form to function properly. Present in prokaryotes as well as eukaryotes. Protein-protein interactionsSection 2 Gene Regulation of Prokaryotic Systems : Section 2 Gene Regulation of Prokaryotic Systems: Common features Prokaryotic genes are polycistron systems , that is, several relevant genes are organized together to form a transcription unit --- operon. The majority of gene regulation is negative. Inducers are used to remove the repression.: Operon is a coordinate unit for the regulation. Transcription initiation is the key point for regulation. Translation can also be regulated. §2.1 Regulation of TranscriptionPowerPoint Presentation: Structure of lac operonPowerPoint Presentation: Metabolism of lactosePowerPoint Presentation: 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 weak promoter , and has a basal expression level. CAP (Catabolite gene activator protein) binding site is at -60 region. CAP is a homodimer with binding ability to DNA and cAMP.PowerPoint Presentation: Glucose inhibits the formation of cAMP. When glucose is present, [cAMP] is lower. Only after glucose is exhausted, [cAMP] becomes higher. The CAP-cAMP complex is formed, and this complex binds to the CAP binding site on lac operon.: When lactose is absent , no lac gene is expressed. Situation 1PowerPoint Presentation: lacI gene has its own promoter, and its expression can produce LacI repressor . The tetrameric Lac repressor binds to the lac operator site O lac . The binding blocks the RNA-pol moving on DNA template, and no lacZ , lacY , and lacA are expressed.PowerPoint Presentation: galactosidase Situation 2 When lactose is present , lacZ, lacY, and lacA genes are expressed.PowerPoint Presentation: 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 gene can be expressed. Galactose , Allolastose and IPTG are referred to as inducer .: 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: When glucose is present, the [cAMP] is low, no CAP-cAMP is formed and the expression of the lac operon is still low. Situation 3: When glucose is absent and lactose is present, the CAP-cAMP complex binds to the CAP site to activate the lac gene. Situation 4: Coordinate expression No glucose Glucose: §2.2 Transcription Attenuation The trp operon is one of the constitutive genes expressed at the basal level. The structural gene of trp operon encodes 5 enzymes used for the synthesis of Trp. Trp operon: 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 Trp. The complex binds to the operator , blocking the synthesis of Trp.: Trp operon: Attenuation mechanism In addition to the repressor regulation, trp gene has a fine tuning mechanism called attenuation. The trp operon is regulated using attenuation mechanism at the translation level.: Leader sequence: Possible hairpins 1/2 and 3/4 hairpin structure 2/3 hairpin structure: High Trp concentration: Low Trp concentration: Under the normal conditions, the LexA gene expressed to repr essor proteins that bind to promoters of other genes and block their expressions. Once the repressors are degraded, the repressed genes will be expressed. At the basal level, the normal cell contains about 1000 copies of RecA protein . §2.3 Protein Degradation: SOS response: LexA digestion: When DNA is extensively damaged, DNA replication is halted and the number of ssDNA gaps increases. The RecA protein binds to this damaged ssDNA, which activates the protein’s coprotease activity. While bound to ssDNA, the RecA protein facilitates the cleavage of LexA repressor as well as the inactivation of the LexA repressor.: §2.4 Genetic Recombination: An RNA, with sequence complementary to a specific RNA transcript or mRNA, whose binding prevents processing of the transcript or translation of the mRNA. A ntisense RNA: A ntisense RNASection 3 Regulation of Eukaryotic Transcription : Section 3 Regulation of Eukaryotic Transcription: Structural features Large genome : 3 x 10 9 bps, 35 000 genes Monocistron Repeated sequences : different lengths and different frequencies. Often inverted repeats Splite genes : separated by introns and exons alternatively: Regulation features 1. RNA-pol: 3 forms (I, II, and III) for different RNAs 2. Changes of chromosomal structure Hypersensitive site Base modification Isomer-conversion Histone changes: 3. Positive regulation 4. Transcription and translation are separated 5. Post-transcriptional modification 6. 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 box,: Sequence: TATA AAA 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 boxPowerPoint Presentation: Sequence: GC CAAT Location: ~ -70 bp Function: It is the binding site for CTF1 (CAAT-binding transcription factor) and C/EBP (enhancer binding protein). CAAT box: Sequence: GGGCGG Location: -30 ~ -110 bp Function: It is the binding site for a protein called Sp1. GC boxPowerPoint Presentation: It is a DNA sequence that can determine the temporal and spatial specificities of expression and increase the promoter activity. 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 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: Promoter and regulatory proteins: General 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.: Transcription repressorPowerPoint Presentation: §3.3 DNA-protein interactions Regulatory proteins have discrete DNA-binding domains of particular structure, i.e., binding motif. The AA side chains of regulatory proteins interact with bases of DNA through H bonds .: Yeast activator protein GCN4 Leucine zipperPowerPoint Presentation: Zinc fingerPowerPoint Presentation: Steroid hormone receptor: Mouse regulatory protein Zif268 Zinc finger: Helix-loop-helix: Helix-loop-helix Human transcription factor MAX: Helix-turn-helix Lac repressor: Helix-turn-helix Trp repressor You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.