BIO 344 1st Edition Lecture 6 Outline of Last Lecture I. Gene Regulationa. Lac operon as an exampleII. Lac operon-- Metabolizing lactosea. Complementation testb. What gene encodes whatIII. Constitutive vs. uninduciblea. What phenotype is seenIV. Cis or trans actingV. What does the repressor repressOutline of Current Lecture I. Lac Repressor Structurea. Homodimerb. Defining ligand binding domainsi. Helix turn helixc. TetramerII. Operator functionIII. DNA binding sites recognition of targeta. Specificityb. AffinityIV. EquilibriumV. Ligand-induced allosterya. cooperativity Current LectureStructure and Function of Lac Repressor and its Operator- trans acting, therefore diffusible- bound to operator- binding site for lac repressor is palindromico this tells us that the binding site is symmetrical, so the lac repressor protein is symmetrical—it’s a homodimer =2 identical proteins boundThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute. Each monomer makes critical contact to DNA in order for it to function- Mutating the operator constitutively would mean the repressor can’t bind; lacZ is turnedon- Modular architectureo Different parts do different things/ bind to different things- Two middle domains of the dimmer mimic flavodoxin- Defining ligand binding domainso Lac repressor binds DNA, allolactose, and itself (to make the dimmer)o Rare lacI-d mutations (d=dominant) Lac repressor d and lac repressor WT lac dominant wins out N-terminus corresponds to the 5’ end of RNA Used lacI-d to map 180 nucleotides of gene (N-terminal 60aa) N-terminus of the lac repressor is the DNA binding domain- Helix-turn-helix motifo Common sequence-specific DNA binding motif in prokaryotic transcription factorso Distance between the recognition helices in the dimmers matches the distance between one major groove and the next, one helix turn away- Alpha helix dimensions match the major groove- Can detect sequence by making hydrogen bonds to groups of nucleotides in the major groove- Mutations in the DNA binding domain are dominanto This is because the lac repressor is a dimmer, both half sites are important and must function to work, which suggests that the protein dimerizes through other domainso If mutant dimerizes with a wild type, wild type can not function and “poisons” ligamers o Dimerization mutation Recessive because mutant will not poison the wild type- If a monomer is mutated and cannot form the dimer, the functioning wild type monomer can still bind to other wild typesOperator Function- If O1 is a lac repressor binding site and O2 and O3 have similar core sequences, do thoseoperators function in vivo?o Mutate the potential operating sites and observe repression activity When mutated, repression is not tightly regulated The other operators must have function in regulation Can we say they are also repressor binding sites?- They are, however we can not say this based on this particular evidence- Lac repressor is actually a dimer of dimmers—tetramero What is the effect of mutant lacI on tetramersDNA binding site recognition of target- Specificity= they must identify their correct binding sites among millions of nucleotides- Affinity= bind effectively to their targets at their in vivo concentrations- They must bind reversibly- Dimmers boost specificity and affinityo A 16 nucleotide recognition sequence needs to then be 2 fold more complex for the dimer to recognize it (greater specificity), and thus 2 fold more hydrogen binding (greater affinity)o The sequence can be read to recognize a locus without separating the strands Can recognize and make hydrogen bonds by recognizing the composition of the major groove for each base pair combination Each base pair in major groove presents a unique site for hydrogen binding- Same at the minor groove- See image on slideso As specificity increases, affinity increases for the sequence’s specific recognizing proteins- Example of the contraryo Helicase is not evolutionarily adapted to be specific, as it’s important to its job to not be specific—zip through quickly without paying attention to sequenceo Other things that recognize DNA nonspecifically are typically positively charged and recognize the sugar phosphate bondEquilibrium Constant vs dissociation constant- Protein + ligand  protein*ligand- Keq= equilibrium constant- Kd= dissociation constant in moles/L- Kon= rate at which two things come together- Koff= rate at which they dissociate at a certain concentration- Kon[protein][ligand]=koff [protein*ligand] at equilibriumo If we increase lac repressor, lac repressor bound to DNA will increaseo If we increase affinity, lac repressor bound to operator will increaseo In a eukaryotic cell, if volume decreases (volume of space in which the protein is working), concentration of the complex will increase- Keq is sensitive to deltaGo High Keq is equivalent to a low KdLigand-induced Allostery (when lactose is present)- Allolactose (inducer—product of beta-galactosidase) binds between flavodoxin domains and distorts helix-turn-helix motif spacing and prevents repressor from binding because affinity is losto Protein conformations often change substantially upon forming complexes- To get it to bind in the presence of allolactose at such a decreased affinity, concentration of the protein would have to be increased greatly- When conformation does change, it must still be stableo Conformation must be uniform—occurs in entire dimero All regulated proteins have at lease 2 stable conformations of about equal energy- Cooperativityo Ligands prefer the same conformationo Ligand binding to one monomer induces a conformational change that facilitates the binding of ligand to a second monomero Oligomeric proteins show sharper titration points in response to binding ligands- Negative regulationo Ligands prefer different