BIOLOGY 305 1st Edition Lecture 23 Outline of Last Lecture I. The Tryptophan OperonII. AttenuationIII. Other Gene Regulation on Biosynthetic PathwaysIV. Vocabulary and Sample QuestionsOutline of Current Lecture I. Introduction to Eukaryotic RegulationII. Transcriptional Regulation of RNA Pol II GenesIII. The Yeast Gal SystemIV. Regulatory Transcription FactorsV. Transcription Factors as ActivatorsVI. Euchromatin, Heterochromatin, and InsulatorsVII. Vocabulary and Sample QuestionsCurrent LectureI. Introduction to Eukaryotic RegulationEukaryotic gene regulation is much more complex than prokaryotic. It must use a variety of methods to regulate/silence their genes at the right location and right time, and to prevent numerous possible expression patterns. Chromatin is a feature that makes eukaryotes restrictive:Goal: 1) Keep genes not being used silent (hence a restrictive structure)2) Use a large number of transcriptional regulators to prevent tons of expression patternsTranscription initiation involves a large complex of proteins: transcription factors help guide RNAPolymerase II to bind to the promoter, a mediator lies at the center and holds everything together, the complex is regulated by regulatory transcription factors and enhancers6 Levels of Regulation:Transcription initiation (primary)  RNA processing, transport, stability  translation efficiency  protein modification/modificationII. Transcriptional Regulation of RNA Pol II Genes trans-acting factors interact with cis-acting elements:3 Cis-acting elements:Promoter – TATA Box (bound to by TBP and TFIID)Promoter Proximal Elements – upstream of transcription start siteEnhancer – distance and orientation independent(enhancers could be almost anywhere, may not be near gene it regulates)Trans-acting proteins (aka transcription factors): diffusible factors bind to cis-acting elements:Transcription factors (that binds to DNA, assists RNA polymerase in initiation):Basal (general) transcription factors – part of the RNA pol II holonezymes that binds the promoter (ex: TFIID)Multiple factors bind to the promoter proximal elements and affect gene expression (but is not mentioned in detail)Regulatory transcription factors – bind enhancers away from promoter regiongives specificity to regulation, regulates smaller subsets of genesThere are multiple regulatory enhancers Transcription factors (that do not bind DNA, binds to proteins bound to DNA):Mediator – very large complex, links transcription factors bound to enhancers and basal transcription apparatus bound to the promoterCoactivators – enhance transcription by binding to transcription factors bound to enhancers, no DNA binding domainCorepressors – like coactivator except represses transcription, no DNA binding domain Two functions for transcription activation:1) Direct interaction with RNA Pol II, with basal transcription factors, or with mediator2) Open chromatin and make more accessible to RNA polymerase IIIII. The Yeast Gal SystemGAL system – comprised of the genes necessary for galactose metabolism, this set of gene work in a really simple way:If galactose is present, genes are turned onIf not, they are turned offThe system is regulated in a coordinated way - each gene has a similar set of enhancersMutants of the yeast GAL system:GAL80 (-/-) mutants – constitutive expression  repression is affectedGAL4 (-/-) mutants – uninducible expression  activation is affectedGAL3 (-/-) mutants – uninducible expression  activation is affectedGAL4 is an activator while Gal 80 is a corepressorGal3 in combination with galactose cause an allosteric transition where this complexes with Gal80 and pulls it off of Gal4. Gal4 is attached to the DNA. When Gal80 dissociates from Gal4, Gal4’s activation domain is activated and transcription is allowed to proceed.In the absence of Gal, Gal80 (co-repressor) binds to Gal4 (activator) and all transcription on chromosome II and chromosome XIII are inactivated.Galactose present  Gal 3 is changed and thus so is its biological activityGal 3 is able to bind Gal 8In the presence of Galactose, Gal80 dissociates and is no longer able to bind to Gal4. Gal4 still stays bound to DNA and is able to promote all transcription on chromosome II and chromosomeXIIICAP and Gal4 in Comparison:CAP is also an activator - how is the yeast Gal system different from the prokaryote lac operon?CAP – it is regulated differently: DNA bindingGal4 – Gal4 is always bound to DNA while its regulated step is activity of the activation domainIV. Regulatory Transcription FactorsRegulatory transcription factors have different domains for different purposes: A DNA binding domain, activation domain (sometimes there is a hormone receptor or dimerization domain)Hormone Receptors:Steroids are hormones that are interact with these receptors. They’re ability to diffuse through the cytoplasmic or nuclear domain is notated:a – steroid means that its receptor is in the cytoplasma + steroid means that its receptor is in the nucleusDimerization: many transcription factors act as dimers.Ex: Myc and MaxAs a heterodimer (Max/Max): acts as an activatorAs a homodimer (Myc/Myc): acts as a repressor and has no activation domainV. Transcription Factors as Activators of TranscriptionRecall the two methods of transcription factor regulation:1) Transcription factors make direct contact with basal transcription apparatus2) Opens up chromatin to make it more accessible to the RNA polymerase II holoenzymea) Chromatin remodeling Nucleosomes (protein) slide along DNA in order to expose the promoter to basal factor and remodeling proteinsb) Post-translational histone modificationsOf which there are numerous kinds:Histones contain marked strings of acetylation and methylation that are recognized by other proteinc) Methylations of DNAAn enzyme called DNMT can methylate cytosine, creating CpG dinucleotidesPHO5 locusUnder high phosphate conditions, transcription does not occur:Under low phosphate conditions, if the histones surrounding the TATA box region are acetylated, transcription still does not occur until nucleosome proteins are ejected. Once they are ejected, phosphate proteins facilitate the binding of a chromatin remodeling complex called SWI/SNF. This complex allows transcription to occur:VI. Euchromatin, Heterochromatin, and InsulatorsChromatin modification results in non-uniform chromatin structure in the nucleus. Chromatin packaging density can