BIO 305 1st EditionFinal Exam Study Guide: Lectures 22 - 28Lecture 22Prokaryotic Regulation Part IIWhat does removing tryptophan from a medium look like on a Northern Blot and why? What happens to the Trp operon at low levels? What do attenuation mutants look like? Describe how attenuation works. Describe the similarities and differences between Trp and Lac operons: Both: A repressor protein binds to an operator and prevents transcription initiationDifference: Anabolic paths do not use inducersThe Tryptophan Operon- The genes that encode the enzymes are organized into operons:How is it regulated? 2 ways:1) Global control of operon mRNA expression2) Fine-tuned controlTrpR gene  Trp repressor  allosteric binding of trypthophanTrpR-  continue to express Trp mRNA  continue to produce tryptophan Indicates a second regulation systemAttenuation: happens post-initiation of transcription, is premature termination of transcriptiontrpL- or trpA-  trp mRNA is produced at maximal levels despite Trp presenceL gene  leader sequence at 5’ end of mRNA  leader peptide: 14 amino-acids + 2 TrpA gene  attenuator: long sequence of U’sNo repressor protein + HIGH Trp = engagement ribosome  early terminationNo repressor protein + LOW Trp = Trp codon stalls ribosome  transcription continuesLogic: abundant Trp  translation of amino acid, coupling of transcription and translation in prokaryotes, termination via hairpin-loopTranslation of leader peptide and # of amino acids on it determines attenuation effectiveness2 Conformations of the Trp mRNA Leader:Alternative Sigma Factors – regulation of large sets of genesSigma factor – facilitate RNA Pol binding to DNA at the promoter to initiate transcription Coordinated expression of large sets of unlinked genes located throughout the genome bring about dramatic physiological and morphological changesEx: Sporulation - sequential expression of alternative sigma factors recognize alternative promoter sequences, providing coordinated expression of large numbers of independent operons and unlinked genes in response to dessication or heatEx: Heat-shock – Goal: activation of enzymes that degrade denatured proteins and chaperons (protein refolding). rpoH switches on in response  sigma32 replaces sigma70  heat-shock promoters recognized  t heat-shock genes transcribed (gene expression profiles changed at global levels) Lecture 23Eukaryotic RegulationDescribe how the GAL system is positively and negatively regulated – what effects would mutations on key players have? What are all the ways GAL4 works and interacts with other proteins? What are all the mechanisms of chromatin remodeling? 6 levels of regulation?Big picture: Molecular signals from inside or outside the cell lead to the binding of regulatory proteins to specific DNA sites outside of protein encoding regions while binding modulates transcription rateEukaryote regulation vs. Prokaryotes: Has transcriptional gene regulation and post-transcriptional gene regulation (gene silencing), chromatin restriction, more regulatory proteins,ground state is off, three RNA polymerase, RNA processing, complex RNA Pol IIGoals: 1) Keep genes not being used silent (hence a restrictive structure)2) Use a large number of transcriptional regulators to prevent tons of expression patterns:Cis-Acting: Promoter – TATA Box (bound to by TBP and TFIID)Promoter Proximal Elements - sequence interacting with transcription initiation proteinsEnhancer – distance and orientation independent, transcription factors bind to this cis regulatory sequence in the DNATrans-acting proteins (transcription factors bind to DNA, assist RNA polymerase in initiation):Basal (general) transcription factors – part of the RNA pol II holonezymes that binds the promoter (ex: TFIID)Regulatory transcription factors – bind enhancers away from promoter regiongives specificity to regulation, regulates smaller subsets of genes 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 – indirectly represses transcription, but no DNA binding domain 5 Functional Domains of regulatory proteins: DNA binding site recognition, RNA polymerase interaction, adjacent protein interaction for cooperativity, affects chromatin condensation, sensor of cell conditions2 Ways Gal4 works to regulate gene expression1) Binds to TBP at activation domain, recruit TFIID, RNA Pol II goes  promoter2) Interacts with mediator complex, which interacts with RNA pol II  promoterco-activator – a protein that facilitates gene activation by transcription factor but is not a DNA binding protein nor a part of the transcription machinery itselfYeast GAL system – comprised of the genes necessary for galactose metabolismGAL 1,2,7 and 10 converts galactose into glucose, 3 regulatory enzyme genes:GAL80 – expressed continuously, always tries to repress Gal 4 unless pulled off by Gal 3GAL 3 – activated by GAL, complexes with Gal80 and pulls it off of Gal4 (sensor + inducer)GAL 4 – activates target genes through UAS, always attached to DNA, and has two domains: a DNA-binding domain and an activation domain UAS – upstream activation sequence / GAL4 enhancersMutants of the yeast GAL system:Gal1-, 2-, 7-, 1- or 10-  gene is silent even with GAL presentGAL80-: constitutive expression  repression is affectedGAL4- : uninducible expression  activation is affectedGAL3- : uninducible expression  activation is affectedCAP and Gal4 in Comparison:CAP – it is regulated differently via DNA bindingGal4 – Gal4 is always bound to DNA while its regulated step is activity of the activation domain (red lines are UAS)Hormone Receptors: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 – separable partsEx: Myc and MaxAs a heterodimer (Max/Max): acts as an activatorAs a homodimer (Myc/Myc): acts as a repressor and has no activation domainEukaryotic activators recruit RNA Pol II to gene promoters by 2 mechanisms:1) Activators recruit protein complexes with roles in transcription initiation2) Activators recruit protein that modify chromatin