BIO 344 1st Edition Lecture 7 Outline of Last 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 Outline of Current Lecture I. Integrating glucose and lactose signalsII. Glucose repression is combinatoriala. cAMPb. CAPIII. Generalizing RegulationIV. Generalizing TranscriptionThese 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.a. RNAPV. Stages of Transcript Initiation by RNAPCurrent LectureIntegrating glucose and lactose signals- Prefer glucose as carbon source- Will use lactose but that requires synthesizing the metabolic machinery neededo Costs energyGlucose Repression is Combinatorial- Combinatorial= repressed or activated by lac repressor or CAP depending on presence orabsence of glucose and lactoseo +glucose/+lactose operon off—CAP not boundo +glucose/-lactose operon off—lac repressor bound and CAP not boundo –glucose/-lactose operon off—lac repressor bound- cAMP is the direct effector of flucose repression and binding of CAPo –glucose/+lactose operon on- CAP is a dimero Helix turn helix motifo Identical subunits each with domains with individual functionso CAP binding bends the DNA significantly with cAMP Each monomer of dimer binds to major groove and DNA bends at almost a 90 degree angle Enhances lac repressor binding to the operator- Strong repression requires 2 activatorso Bending allows the repressor bound to O1 to bind to O3 as wellGeneralizing Regulation- Negative/ Inducible gene off o Repressor bound to operatoro Adding ligand will remove repressor and turn the gene on- Negative/ repressible gene offo Molecule binds to repressor to bind to operator to turn the gene offo Remove ligand to remove the repressor to turn the gene on- Positive/ repressible gene ono Ex: CAPo Activator bound to operator, so gene is ono Add ligand to turn the gene off- Positive/ inducible gene ono Molecule bound to activator which is bound to the operator, so gene is ono Removing the ligand removes the activator to turn the gene offGeneral Transcription- Alignment of DNA fragments protected by RNA polymerase (RNAP)- Star site is at +1o Next nucleotide upstream is -1 (no zero)- Consensus sequence is at -35 and -10o Sequence is mostly similar at that position to TTGAC for -35- Not absolutely the same, but generally similaro Spacing between -10 and -35 matters- If we add 5 nucleotides, we would destroy the promoter, as this would change the location of the consensus sequence spacing by a half turn of the helix- Turn of the helix would put them on different faces- If we add 10, the promoter would still work, because spacing would be wrong, but the helix would turn completely, keeping them on the same face- RNAP holoenzymeo Core= alpha, beta, beta’, w- Catalytic, not specifico Holoenzyme= sigma, alpha, beta, beta’, w- Catalytic and specific- Sigma factor adds specificity for recognizing the promoter- Different sigma factors recognize different promoter sequences- Sigma 70 recognizes -10 and -35 consensus sequence so RNAP canbind to the upelement- Kon is greater for a perfect consensus sequence (greater promoter strength)- That promoter will be favored by RNAP for transcriptiono Regulates extent of gene expression because more of that gene will be transcribed than the other- If RNAP concentration is increased to saturation, will drive to express both geneso Regulation will decreaseTranscription Initiation- Stages of Transcription initiation by RNAPo Closed complex= RNAP bound to DNAo Open complex= (irreversible) at -10, open strands, DNA will start synthesis of RNAo Abortive initiation= small sequences will be synthesized and transcription will stop—this repeats several times until elongationo Elongation complex= escapes promoter and can start elongating- Sigma factor melts the -10 region by competing with the template for binding nontemplate in order to dissociate the template from the nontemplate to open strands for transcription - Kb is a function of the promotero Dictated largely by -35o Affinity for RNAP promoter- Mutations at different nucleotides of -35 or -10 can increase or decrease function depending on if the mutation increases or decreases promoter strength (consensus sequence more similar or different)- Can evolve a better promoter by adding an up element- CAP adds an extra binding surface and will recruit
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