Eukaryotic Transcription II

StudyBlue printing of Eukaryotic Transcription II html, body, div, span, applet, object, iframe, h1, h2, h3, h4, h5, h6, p, blockquote, pre, a, abbr, acronym, address, big, cite, code, del, dfn, em, font, img, ins, kbd, q, s, samp, small, strike, strong, sub, sup, tt, var, b, u, i, center, fieldset, form, label, legend, table, caption, tbody, tfoot, thead, tr, th, td { margin: 0; padding: 0; border: 0; outline: 0; font-size: 100%; background: transparent; } body { line-height: 1; } blockquote, q { quotes: none; } blockquote:before, blockquote:after, q:before, q:after { content: ''; content: none; } /* remember to define focus styles! */ :focus { outline: 0; } /* remember to highlight inserts somehow! */ ins { text-decoration: none; } del { text-decoration: line-through; } /* tables still need 'cellspacing="0"' in the markup */ table { border-collapse: collapse; border-spacing: 0; } /* end RESET */ .header { min-width:800px; } .logo { padding:6px 20px 2px 20px; margin:0; font-size:25px; font-weight:bold; color:#808285; position:relative; border-bottom: 1px solid #c5c5c5; } .logo-blue { color:#70adc4; } .logo-desc { font-weight:normal; font-size:19px; color:#cccccc; margin-top:50px; position:absolute; display: none; } .back-button { position:absolute; top:20px; right:20px; font-size:13px; line-height:25px; color:rgb(0,175,225); font-weight:normal; } .back-button a { color:rgb(0,175,225); } .instructions { padding:0; margin:0; width:100%; position:relative; color:rgb(100,100,100); } .step-holder { border-left:1px solid #ededed; margin-left:20px; } .steps { padding:15px 0; float:left; width:24%; border-right:1px solid #ededed; text-align:center; } .steps-01 { } .steps-02 { } .steps-03 { } .steps-04 { } .label { padding:5px 10px; } .print-button { } .print-button a { background-color:rgb(0,175,225); color:white; line-height: 19px; padding:9px 8px 5px 30px; font-size:14px; text-decoration:none; background-image: url(images/printer.png); background-repeat: no-repeat; background-position: 7px 50%; -moz-border-radius: 5px; -webkit-border-radius: 5px; } .print-button a:hover { background-color:black; } .theNote .content { width: 8.0in !important; margin: 5px auto; padding:20px; background-color:white; } .theNote .header { border-bottom: 1px dashed #C8C8C8; font-size: 17px; padding: 0 0 10px; line-height: 19px; color: #00ADE1; min-width:500px; } .theNote .body { font-size: 14px; line-height: 19px; padding: 10px 0; } .theNote{ padding:6px 0; clear:both; background-color: rgb(200,200,200); } .theNote h3{ color: rgb(100,100,100); } .theNote h1, .theNote h3{ background-color:white; padding:2px 20px; width:8.0in !important; margin: 0 auto; font-size: 15px; } .theNote h1{ padding-top: 10px; font-size: 15px; } .theNote h1:first-child{ font-size: 20px; } .theNote h3 { font-size: 14px; font-weight: normal; } #options { border: 3px double #ccc; padding: 5px 12px; margin: 10px 50px 10px 20px; float: left; } #info { border-top: 1px solid #ccc; padding-top: 5px; font-style: italic; } li { margin: 5px 10px 5px 25px; } ul li { list-style: disc; } ol li { list-style: decimal; } img { border: 0; } table { clear: both; width: 100%; border: 1px solid #c5c5c5; border-width: 1px 0; margin: 0; page-break-after: always; } table#page { page-break-after: auto; } td { text-align: center; font-size: 12px; border-bottom: 1px dashed #c5c5c5; height: 1.75in; width: 50%; padding-left: 15px; } .leftside { border-right: 1px solid #cccccc; padding: 0 15px 0 0; } .bottom td { border-bottom: none; } .clearfix { clear:both; line-height:1px; height:1px; } img { max-width:80%; max-height:150px; margin:20px; } @media print {.header { display: none; } .content .header{ display:inherit; } table { border: 1px dashed #bbb; border-width: 1px 0; } .theNote{ background-color:white; } } Eukaryotic Transcription II General facts TFII factors are required for basal transcription Higher levels of transcription are activated by transcription activators Transcription factors - transcription regulators that bind to specific DNA sequences to suppress or activate transcription. Most DNA encodes transcription factors Transcription factors can control more than one gene Transcription often bind to DNA as dimers Increased local concentration of DNA-binding motifs Decreases energy needed for binding DNA Increases diversity Structure of transcription factors Transcription factors have two major domains DNA binding motif - allows protein to bind to DNA Activating motif - allows protein to interact with other proteins, to activate or suppress transcription Zinc finger Bind Zinc 2+ factors Contain regularly spaced cysteines and histidines One zinc finger protein may have more than one zinc finger. Usually composed of atleast one beta strand and one alpha helix May bind as monomer or dimer GR (Glucocorticoid receptor) Family of zinc finger transcription factors called nuclear receptors Inactive in the cytoplasm, but active in the nucleus GR is normally locked by HSP (Heat-shock proteins) Cortisol (Glucocorticoid) disrupts the binding of GR to HSP, and GR travels in to the nucleus and binds to DNA, suppressing or activating transcription Homeodomain (HD) Homeodomain-containing transcription factors HD is a ~60 residue containing the helix-turn-helix DNA sequence encoding the homeodomain is called the homeobox Most HD has weak DNA-binding activity, so need other proteins to help bind to DNA Homeodic genes - genes, when mutated, will transform one body part to another Ex. Fly Ey gene Master control of expression of 2500 genes needed for eye development Mutation here causes eyes to pop up in random places Ex. SPD (Human Synpolydactyly) HOX Protein encodes 38 proteins, mutations in this HOX gene causes SPD bZIP Basic Leucine Zipper proteins Contain long stretches of repeating leucines Allows for hydrophobic interactions between zippers Dimers bind to DNA like tongs/forceps, stabilizing DNA very well (called coiled-coil structure) Ex. AP1 Transcription factor composed of two bZIP proteins, Jun and Fos Nuclear receptors inhibits dimers of Fos and Jun by JNK (a protein kinase) AP1 controlled by acetylCoA JNK phosphorylates Jun and binds to Fos to become active bHLH Structurally similar to bacterial Helix-turn-helix, as in the LacI Dimer, positively chared basic region acts as DNA-binding Ex. Myc and Max first discovered bHLH, also called bHLH-ZIP because they also have a ZIP domain Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells c-Myc gene can be mutated and become overexpressed (gain of function), causing cancer, becomes worse when ras D is also mutated Proto-oncogene Normal gene that can cause cancer Notated by c-[GENE] like c-Jun, c-Fos Oncogene Gene, most often a transcription factor, when mutated, causes cancer Notated by [GENE] like Jun, Fos Tumor suppressing genes Loss of function, suppression of cell proliferation becomes broken and cells grow without inhibition p 53 is the best known tumor suppresor gene. May act as a suppressor or activator affects all three RNA polymerases Mechanisms of transcription factors 1. Transcription activators may help recruit TFIID to the promoter, or recruit RNA polymerase to the TFIID/promoter complex Both mechanisms stabilize binding of RNA polymerase, resulting in increased transcription Experiment Effector plasmid, encodes the LexA-GAL11, which is a fusion protein of the DNA-binding domain of a bacterial transcription repressor LexA and a yeast TFII general transcription factor Reporter plasmid contains LaxA-LacZ reporter gene, controlled by promoter and LexA-binding site Transform both plasmids and test lacZ transcription by beta-galactosidase activity Result: LexA-GAL11 chimeric protein can activate lacZ expression Conclusion: A transcription factor can bind to DNA and help bring RNAP to the promoter to activate transcription 2. Different domains of a transcription factor can function independently Can still function even when detached from other parts of the protein 3. Transcription activators usually bind to enhancers from far away to the promoter, but relay its action by DNA looping Common mechanisms: Sliding, Looping, Tracking 4. One gene is usually simultaneously regulated by multiple transcription activators and repressors to achieve optimal level of transcription. This is called combinatorial regulation Enhanceosome - DNA protein complex of multiple trnascription factors associated with the enhancer. Helps integrate combinatorial effects of all transcription factors 5. Depending on proteins it interacts with, a transcription activator may act as a transcription repressor under different conditions Unknown mechanism 6. The function of a transcription activator may be regulated by other proteins, such as mediators, insulators, and co-activators Two problems with transcription activators - How do enhancers affect more than one gene? and what happens when an activator or a repressor is no longer needed? Insulators - a protein that suppresses the activity of an enhancer by preventing DNA looping or preventing the spreading of chromatin condensation May also work by binding between an enhancer and promoter to prevent sliding May also work by looping out the enhancer, so the activator cannot form a loop with the promoter Regulation of transcription factors 1. Mediator: co-activator Multiple sub-unit complex that binds to the preinitiation complex Mediator is a co-activator required for activator dependent sitmulation of transcription 2. Protein phosphorylation The more phosphates on RNAPII, the more active it becomes 3. UPS and Protein degradation The Ubiquitination/26S proteasome system Adding ubq tags on lysine residues will mark the protein to be degraded by 26S proteasome E1 - (Ubq activating enzyme) - activates the Ubq peptides E2 - (Ubq conjugating enzyme - catalyzes the Ubq attachment reaction E3 - (Ubq-ligase) - recognizes and brings substrates to E2 P 53 can bind to HDM2 ubq E3 ligase HDM2 targets P 53 and can ubq it and degrade it This is the target for cancer therapy because P 53 causes cancer P 53 can be phosphorylated and attach to a Pin1 transcription coactivator. This disrupts the P 53 -HDM2 bond. Without HDM2, P53 can once again activate transcription 4. Acetylation and Methylation Acetylation Adds acetyl groups (CH 3 O groups) to proteins, on Lysine residues or N-terminals, catalyzed by N-alpha-acetyltransferases. Some can also acetylate the epsilon group on the N-terminal by a acetyltransferase. Arginine is usually not acetylated Methylation Adds methyl groups at arginine or lysine by methyltransferases Transcription and signal transduction Cellular responses to internal and external signals Complicated signal transduction cascades can amplify signals or provide interactions of different signals, creating a signal transduction network Transduction usually controls transcription by controlling the activity of transcription factors Histone acetylation High levels of cAMP -> PKA in nucleus -> phosphorylates CREB (cAMP response element-binding protein -> binds to CBP (CREB-binding protein) -> CBP contacts basal complex to activate transcription CBP is a histone acetyltransferase (HAT) Histone methylation Nuclear receptors (NR) move into the nucleus and bind to Hormone-response elements (HRE) DNA sequences without the hormone, but cannot recruit the basal complex without co-activators When NR binds to hormone, it interacts with a co-activator, SRC (Steroid receptor co-activator). SRC recruits CBP (HAT) and CARM1 (HMT) CBP acetylates histones to activate transcription. CARM1 methylates histones to boost transcription ALSO, CARM1 also methylates the arginine residue of CBP to prevent it from interacting with CREB, so CBP cannot accidentally activate cAMP-PKA regulated genes Methylation can both repress and activate transcription Crosstalk - intentional, desired signal crossover Multiple pathways lead to the same protein that activates transcription. Signal transductions work together to get the correct amount of expression from crosstalk Regulation of transcription factors Activity of transcription may be regulated by interacting with other proteins or by chemical modifications Protein level of transcription factors can be regulated by ubiquitination/26S proteasome pathway Transcription is regulated by the chromatic structure Recruitment of histone-modifying enzymes to the promoter DNA is major mechanism underlying function of transcription factors Cellular signal transduction networks regulate the activity of transcription factors